New, sensitive thin-layer chromatographic-high-performance liquid chromatographic method for detection of trichothecene mycotoxins

Diphenylindenone sulphonyl (Dis) esters of trichothecene mycotoxins when sprayed with sodium methoxide showed fluorescent spots on a thin layer of silica gel when viewed under long-wavelength UV light. The detection limit for trichothecene esters in thin-layer chromatography (TLC) was 20-25 ng per spot for T-2 toxin, HT-2 toxin, diacetoxyscirpenol, T-2 triol, T-2 tetraol and iso-HT-2 toxin. A quantitative high-performance liquid chromatographic (HPLC) analysis of Dis-trichothecene esters was also developed using UV detection at 278 nm. The detection limit for the above esters varied between 30 and 50 ng per injection. This sensitive TLC-HPLC method is very useful for in vivo pharmacokinetic analyses of trichothecenes.     

Analysis of ochratoxin A in milk after direct immunoaffinity column clean-up by high-performance liquid chromatography with fluorescence detection

The present work describes a new analytical method for direct immunoaffinity column clean-up of ochratoxin A (OTA) in milk samples followed by determination of the toxin using high-performance liquid chromatography with fluorescence detection (HPLC-FD). Two different immunoaffinity cartridges (IAC) were investigated, and Ochraprep columns were chosen because they showed the best results. An average recovery of 89.8% and a mean RSD of 5.8% for artificially contaminated cow's milk in the range of 5-100 ng/L were attained. The calculated limit of detection (LOD) and limit of quantitation (LOQ) were as low as 0.5 and 5 ng/L, respectively. This new easy and fast method avoids a previous liquid-liquid extraction step and therefore the use of toxic chlorinated solvents. Chromatograms of the final extracts were clean and OTA could be easily detected at a retention time of 8.4 min without interferences. To assess the presence of the toxin in cow's milk eight samples of skimmed and four samples of whole milk were analysed and OTA was not detected over the established detection limit.     

Ionspray mass spectrometry of marine toxins. IV. Determination of diarrhetic shellfish poisoning toxins in mussel tissue by liquid chromatography/mass spectrometry.

An improved liquid chromatographic/mass spectrometric (LC/MS) method utilizing gradient elution and ion-spray ionization is described for the sensitive determination of okadaic acid and dinophysistoxin-1, the principal toxins implicated in cases of diarrhetic shellfish poisoning. The method was used to confirm the presence of both toxins, together with a recently identified isomer of okadaic acid, dinophysistoxin-2, in various samples of cultivated blue mussels (Mytilus edulis) from Canadian and European waters. The method provided a mass detection limit of 0.4 ng for each toxin, thus allowing detection of 40 ng per g of whole mussel tissue (or approximately 10 ng/g if only the digestive glands were used in the assay). Quantitative results obtained by LC/MS were in good agreement with those obtained by derivatization and high-performance liquid chromatography with fluorescence detection.     

高效液相色谱法与酶联免疫试剂盒测定海水中痕量氯霉素

详细报道了海水样品中氯霉素的高效液相色谱检测方法及酶联免疫试剂盒检测方法。应用高效液相色谱法测定海水中氯霉素,方法的线性范围为5～50ng,检出限为0．064ng,回收率为96．2％-118．4％,相对标准偏差小于8．9％。酶联免疫试剂盒测定海水中的氯霉素背景值偏高,在加标浓度为10ng／mL时回收率为101．0％,相对标准偏差为8．32％。     

Determination of ochratoxin A in domestic and imported beers in italy by immunoaffinity clean-up and liquid chromatography

A method first developed to quantify ochratoxin A in wine has been applied to the analysis of domestic and imported beers in Italy. The method uses commercial immunoaffinity columns for clean-up and high-performance liquid chromatography for quantification of the toxin. Beer was degassed, then diluted with a polyethylene glycol-sodium hydrogencarbonate solution and applied to an OchraTest immunoaffinity column. Ochratoxin A was eluted from the immunoaffinity column with methanol and quantified by reversed-phase HPLC with fluorometric detector. Average recoveries of ochratoxin A from blank beer spiked at levels from 0.04 to 1.0 ng/ml ranged from 93.8% to 100.4%, with relative standard deviations between 3.3% and 5.7%. The detection limit was 0.01 ng/ml based on a signal-to-noise ratio of 3:1. The analysis of 61 samples of domestic (10) and imported (51) beers showed ochratoxin A levels ranging from <0.01 to 0.135 ng/ml with an incidence of contamination of 50% and no substantial difference between strong and pale beers.     

免疫亲和柱净化/柱前衍生化-高效液相色谱荧光检测法测定粮谷中的T-2毒素

建立了免疫亲和柱净化/柱前衍生化-高效液相色谱荧光检测器测定粮谷中T-2毒素含量的方法。样品经甲醇-水(体积比为80∶20)混合溶剂提取,通过免疫亲和柱(IAC)净化,以氰酸蒽(1-AN)为衍生化试剂、4-二甲基氨基吡啶(DMAP)为催化剂进行衍生,以ZORBAX Eclipse XDB-C18 柱为分离柱,乙腈-水(体积比为80∶20)为流动相进行高效液相色谱分离及荧光检测，荧光检测的激发波长为381 nm，发射波长为470 nm。T-2毒素的质量浓度为0.01~1.5 mg/L时与峰高呈良好的线性,相关系数为0.9985。在0.01~1.5 μg/g添加水平下，回收率为79.7%~94.5%,相对标准偏差小于7%；检出限（S/N＝3）为0.01 μg/g。该方法净化效果好,灵敏度高,操作简便快速。     

Preparative method for isolating alpha-zearalenol and zearalenone using extracting disk

A liquid chromatographic method is described for the determination of zearalenol and zearalenone in corn. Zearalenol and zearalenone are extracted from corn with methanol-water (1 + 1) and cleaned up using a solid-phase extraction (SPE) disk, separated on a reversed-phase analytical column, and detected with a fluorescence detector. The SPE disk concentrated and cleanly separated zearalenol and zearalenone from sample interferences. Standard calibration curves for zearalenol and zearalenone for the concentration range 25-500 ng/mL were linear. The small extract disk had a column capacity equivalent to 1 g extracted corn. Zearalenol and zearalenone were added at levels ranging from 10 to 2000 ng/g to a control sample that contained no detectable levels of zearalenol and zearalenone. Both toxins were recovered from spiked samples at 106.3 and 103.8%, with coefficients of variation of 7.6 and 13.0%, respectively. The method has an estimated reliable limit of detection and limit of quantitation around 10 and 40 ng/g for each toxin, respectively.     

Direct injection of urine on a high-performance liquid chromatographic column-switching system for determination of delta 9-tetrahydrocannabinol-11-oic acid with both ultraviolet and electrochemical detection

A method is presented for determination of delta 9-tetrahydrocannabinol-11-oic acid in urine by means of a fully automated liquid chromatographic system. Aliquots (200 microliter) of hydrolysed urine from prison inmates were directly injected onto a pre-column, followed by chromatography on two columns with different selectivity: CN and C8 columns. To obtain both greater selectivity and a low detection limit a twin-detector principle was used, consisting of both ultraviolet and electrochemical detection. Urine samples found to be positive with the EMIT cannabinoid were analysed, and the results were compared with those obtained from a well established gas chromatographic-mass spectrometric method. The precision of the method was 2.8% at a mean concentration of 85 ng/ml and 13.4% for 6 ng/ml of the acid. The detection limit was below 5 ng/ml.     

Enzyme-linked immunosorbent assay for the determination of T-2 toxin in cereals and feedstuff

A reliable enzyme-linked immunosorbent assay method was developed for the assay of T-2 toxin in cereals and feedstuff. A hapten of the T-2 toxin was synthesized, and a polyclonal antibody with high affinity and specificity was obtained after immunization of rabbits. Compared to the other ELISA methods, the assay is simple, rapid and affordable. The concentration of T-2 causing 15% inhibition is 0.01?±?0.001 ng mL^?1, which makes the method more sensitive than others. The cross-reactivity against other mycotoxins is low, except for the HT-2 toxin. Sample extraction was achieved within 3 min. The recoveries from samples including barley, wheat, corn, oat, rice and feedstuff were between 75% and 102%, and the detection limit for T-2 toxin was lower than 4 ng g^?1. The method was validated by high-performance liquid chromatography with tandem mass spectrometry detection.     

Determination of type A trichothecenes by high-performance liquid chromatography with coumarin-3-carbonyl chloride derivatisation and fluorescence detection

A method for the analysis of type A trichothecenes T-2 toxin, HT-2 toxin, neosolaniol and diacetoxyscirpenol by high-performance liquid chromatography with fluorescence detection using coumarin-3-carbonyl chloride has been developed. Different parameters concerning the analytical procedure such as stability of both the reagent and derivatised analytes, time and temperature of the derivatisation reaction, were studied and optimised. Three different clean-up procedures (solid-phase extraction with silica gel or C-18 cartridges, and liquid–liquid partition between toluene and dihydrogen phosphate buffer) were tested in order to remove the excess reagent peaks. The last procedure gave the best results when the buffer pH was 3–5.5, and is therefore recommended. Separations were performed on a stainless steel LiChrospher 100 C-18 reversed-phase column with pre-column of the same phase. The mobile phase was acetonitrile/water (65:35, v/v) containing 0.75% acetic acid at a flow-rate of 1.0 ml/min. The proposed method provides good separation between the four trichothecenes and good reproducibility (RSD of calibration standards <5%). The limits of detection of the studied trichothecenes at a signal-to-noise ratio of 3:1, with an injection volume of 20 μl were 10 ng/g sample for T-2 toxin and about 15 ng/g sample for the remaining mycotoxins. The calibration curve was linear between 10 and 2000 ng for the four trichothecenes assayed. The method was applied to the analysis of these mycotoxins in fungal cultures (corn and rice) of Fusarium sporotrichioides, and is also perfectly suitable for the quantification of type A trichothecenes in contaminated cereals.     

Determination of Oltipraz in serum by high-performance liquid chromatography with optical absorbance and mass spectrometric detection.

Three methods have been developed for the analysis of Oltipraz in serum. A method suitable for routine use employs spiking with a homologous internal standard, off-line solid-phase extraction, high-performance liquid chromatographic separation, and optical absorbance detection at 450 nm. Method detection limit is about 1 ng/ml. A second method, less susceptible to bias from co-eluting interferences, uses a stable isotope-labeled internal standard, similar extraction and separation, and detection by thermospray mass spectrometry. Method detection limit is about 0.2 ng/ml. A third method was developed which can be used without specially synthesized internal standards. It uses on-line solid-phase extraction, with quantification by comparison with external standards. Method detection limit is about 3 ng/ml. Good agreement was observed between these methods and with similar and different methods run in other laboratories. Calibration curves were linear over the entire range which was investigated, i.e., up to 500 ng/ml. Coefficients of variation were similar for all three methods, being about 5%.     

Simultaneous separation and determination of 20 potential adulterant antigout and antiosteoporosis pharmaceutical compounds in herbal food products using LC with electrospray ionization MS/MS and LC with quadrupole‐time‐of‐flight MS

An analytical method for the simultaneous and reliable determination of 20 antigout and antiosteoporosis pharmaceutical compounds in adulterated health food products was developed using liquid chromatography with electrospray ionization tandem mass spectrometry and liquid chromatography with quadrupole‐time‐of‐flight mass spectrometry. The method was validated through the determination of specificity, linearity, limit of detection, and limit of quantification, method detection limit, method quantitation limit, precision, accuracy, recovery, and stability. The matrix effect was also determined. The validation results of the developed method are as follows: for solid and liquid blank samples, limits of detection ranged from 0.05 to 5.00 ng/mL and limits of quantification ranged from 0.15 to 15.00 ng/mL. Linearity was acceptable, and the correlation coefficients (R²) were ≥0.99 for all target compounds. Both intra and interday precision were less than 9.16% RSD, and accuracies ranged from 95.31 to 116.68%. Mean recoveries for different types of dietary supplements classified as powders, liquids, tablets, and capsules were found to be 80.81 to 117.62% with less than 15.00% relative standard deviation. The stability of the standard mixture solution was less than 11.72% relative standard deviation after 48 h. By the proposed method, the presence of dexamethasone was determined in seized herbal food products at concentrations that ranged from 126 to 215 µg/g.     

Determination of organophosphate pesticides in sea and surface water with ultrasound-assisted dispersive liquid–liquid micro-extraction coupled to GC-MS/MS analysis

A sensitive enrichment method for the detection and quantification of 20 organophosphate pesticides in sea water and surface water was improved and validated. The compounds were extracted using ultrasound-assisted dispersive liquid–liquid microextraction and were analysed with an optimised Gas Chromatography - Tandem Quadrupole Mass Spectrometry (GC-MS/MS) method. Calibrations and validations were conducted for spiked local pond and Baltic Sea water. Recoveries of the organophosphate compounds from the different matrices ranged from about 25% to 70% and from 30% to 130% for pond and sea water, respectively. Obtained limit of detection (LoD) for both matrices was about 3 ng L^?1 and the limit of quantification (k = 3) was 12 and 7 ng L^?1 for pond and sea water, respectively. Valid extractability at the LoD level could be proven.     

Identification and quantification of 34 drugs and toxic compounds in blood,urine, and gastric content using liquid chromatography with tandem mass spectrometry

A liquid chromatography with tandem mass spectrometry method was developed for the simultaneous screening of 34 drugs and poisons in forensic cases. Blood (0.5 mL, diluted 1:1 with water) or 1.0 mL of urine was purified by solid‐phase extraction. Gastric contents (diluted 1:1 with water) were treated with acetonitrile, centrifuged, and supernatant injected. Detection was achieved using a Waters Alliance 2695/Quattro Premier XE liquid chromatography tandem mass spectrometry system equipped with electrospray ionization, operated in the multiple reaction monitoring modes. The method was validated for accuracy, precision, linearity, and recovery. The absolute recovery of drugs and toxic compounds in blood was greater than 51% with the limit of detection in the range of 0.02–20 ng/mL. The absolute recovery of drugs and toxic compounds in urine was greater than 61% with limit of detection in the range of 0.01–10 ng/mL. The matrix effect of drugs and toxic compounds in urine was 65–117% and 67–121% in blood. The limit of detection of drugs and toxic compounds in gastric content samples were in the range of 0.05–20 ng/mL. This method was applied to the routine analysis of drugs and toxic compounds in postmortem blood, urine, and gastric content samples. The method was applied to actual forensic cases with examples given.     

固相萃取–HPLC–ICP/MS法测定水中的烷基汞

建立了固相萃取、高效液相色谱与电感耦合等离子体质谱联用技术测定水中烷基汞的方法。对固相萃取、高效液相色谱和电感耦合等离子体质谱的实验条件进行了优化。在优化条件下,甲基汞和乙基汞的检出限分别为0.5,0.7 ng/L。对地表水、工业废水和生活污水3种水样分别加标10.0,50.0 ng/L进行测定,测定结果的相对标准偏差为3.3%~9.6%(n=6),加标回收率在81.0%~103.0%之间。该方法灵敏度高,实用性强。     

Determination of ochratoxin A in wine by means of immunoaffinity column clean-up and high-performance liquid chromatography

A new and accurate method to quantify ochratoxin A (OA) in table wine has been developed. The method uses commercial immunoaffinity columns for clean-up and high-performance liquid chromatography (HPLC) with fluorescence detection for quantification of the toxin. Wine was diluted with a solution containing 1% polyethylene glycol (PEG 8000) and 5% sodium hydrogencarbonate, filtered and applied to an OchraTest immunoaffinity column. The column was washed with a solution containing sodium chloride (2.5%) and sodium hydrogencarbonate (0.5%) followed by water. OA was eluted with methanol and quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 333 nm, emission wavelength 460 nm) using acetonitrile-water-acetic acid (99:99:2) as mobile phase. Average recoveries of OA from white, rosé and red wine samples spiked at levels from 0.04 to 10 ng/ml ranged from 88% to 103%, with relative standard deviations (RSDs) between 0.2 and 9.7%. Detection limit was 0.01 ng/ml based on a signal-to-noise ratio of 3:1. The method was applied successfully to 56 samples of red (38), rosé (8), white (9) and dessert (1) wine. The levels of OA ranged from <0.01 to 7.6 ng/ml with red wines more contaminated than rosé and white wines. A good correlation (r=0.987) was found by comparative analysis of 20 naturally contaminated samples using this method and the method of Zimmerli and Dick with better recoveries of OA and better performances for the new method. Several advantages of this method with respect to the actually available methods have been pointed out, with particular reference to red wine which appears to be the most difficult to analyze.     

Improved high performance liquid chromatographic determination of ginsenosides in Panax ginseng-based pharmaceuticals using a diol column

A reversed-phase high-performance liquid chromatographic assay for the simultaneous quantitative determination of seven ginsenosides, Rb(1), Rb(2), Rc, Rd, Rg(1), Re and Rf in pharmaceutical preparations is described. Chromatographic separation was achieved in less than 20 min using a 250 x 4 mm Lichrospher, 5 microm, 100 A diol column with detection at 203 nm. The method was validated over the range of 2.5-20 ng/microL using a 20 microL sample volume. The average accuracy at five concentrations was 90-100%, and the within-day and between-day precision ranged from 1 to 7% expressed as coefficient of variation. The detection limit and the quantitation limit of the method were 20 and 50 ng injected for each ginsenoside, respectively.     

Hydrophilic interaction liquid chromatography/mass spectrometry for determination of domoic acid in Adriatic shellfish

This paper describes a new method for sensitive, specific and direct determination of domoic acid (DA), the causative toxin of amnesic shellfish poisoning (ASP) syndrome, in shellfish. It is based on combination of hydrophilic interaction liquid chromatography with mass spectrometry (HILIC/MS). The high percentage of organic modifier in the mobile phase and the omission of ion-pairing reagents, both favoured in HILIC, result in enhanced detection limits with MS detection. The new method was set up either on an ionspray ion trap MS instrument operating in MS and MS/MS scanning acquisition modes, or on a turboionspray triple-quadrupole MS system operating in selected ion monitoring (SIM) and multiple reaction monitoring (MRM) acquisition modes. Positive and negative ion experiments were performed. MRM experiments are recommended for screening contaminated shellfish tissue and for quantitative analyses due to highest sensitivity and selectivity. The minimum detection levels for the toxin in tissue were found to be 63 and 190 ng/g in positive and negative MRM experiments, respectively, which are well below the regulatory limit for DA in tissue (20 microg/g). Application to shellfish samples collected in the Adriatic Sea (Italy) in the period 2000-2004 demonstrated for the first time in Italy the presence of DA as a new toxin that has entered the Adriatic Mytilus galloprovincialis toxin profile.     

流动注射在线液—液萃取原子吸收光谱法测定生物样品中的铅

本文利用所设计的一种新型流动注射液－液萃取重力分相器，建立了流动注射液溶剂萃取原子吸收光谱法测定铅的新方法，详细地研究了流动注射在线萃取的实验条件及流路系统，方法的精密度和检出限分别为２．５％（ｎ＝１１）和２．８μｇ／Ｌ（ｋ＝３）。用拟定的方法测定了峰蛹，粮食等生物样品中的铅，结果与参考值相吻合。     

Improved high-performance liquid chromatographic determination with amperometric detection of alpha-amanitin in human plasma based on its voltammetric study

A sensitive and simple method is described for the selective determination in human plasma of alpha-amanitin, the most poisonous and prevalent toxin in the lethal fungi of species Amanita, using high-performance liquid chromatography with amperometric detection. After an extraction of plasma with disposable C18 silica cartridges, the extracts were separated by isocratic reversed-phase chromatography using a macroporous poly(styrene-divinylbenzene) column and a mobile phase of 0.05 M phosphate buffer-acetonitrile (91:9) at the apparent pH of 9.5. Amperometric detection was performed by applying an oxidation potential as low as +350 mV (vs. Ag/AgCl) to a glassy carbon electrode, in a thin-layer flow-cell. The linear range for alpha-amanitin was 3-200 ng/ml, and the relative limit of detection in plasma was 2 ng/ml at a signal-to-noise ratio of 2. The intra-assay precision was evaluated at levels of 10 and 200 ng/ml; the coefficients of variation were 4.5 and 2.6% (n = 5), respectively. Inter-assay coefficients of variation were 6.5 and 4.2% (n = 5) for the same concentrations of toxin. These analytical conditions have been chosen on the basis of a preliminary in batch cyclic voltammetric investigation of alpha-, beta- and gamma-amanitins, which has allowed their oxidation process to be clarified and the pH dependence of their oxidation potentials to be determined. All three amanitins are oxidized at the same potential values, and adsorption onto the electrode surface of both reactant and products was found in all cases. This adsorption did not affect the signal recorded for alpha- and gamma-amanitins at the amperometric detector, and for beta-amanitin a stronger adsorption for the anodic product was found, which leads to a marked positive shift of the potential required for the oxidation of this isomer in the amperometric detector cell.