高效液相色谱法测定贝类中的软骨藻酸

 介绍了用反相高效液相色谱 (RP HPLC)测定贝类中软骨藻酸的方法。样品以V(甲醇 )∶V(水 ) =1∶1的溶液提取 ,经LC SAX强阴离子柱固相萃取净化 ,用RP HPLC定量分析。方法的最小检出限为 0 2 μg/ g ,在 1 0mg/L～ 2 5 0mg/L范围内有良好的线性关系 ,测定结果准确 ,重现性好 ,回收率大于 96 %。     

QuEChERS净化技术结合超高效液相色谱-串联质谱法筛查食用贝类中的3种原多甲藻酸贝类毒素

采用超高效液相色谱-串联质谱(UHPLC-MS/MS)技术,建立了贻贝、牡蛎、蚌类、扇贝等食用贝类及其制品中3种天然形式的原多甲藻酸(azaspiracid-1, azaspiracid-2, azaspiracid-3)贝类毒素的检测方法。样品采用乙腈-水(85:15, v/v)混合液均质提取,应用QuEChERS技术净化,以0.2 μm微孔滤膜过滤,在乙腈-水(含5 mmol/L醋酸铵和0.1%甲酸)体系下进行梯度洗脱,并在ZORBAX Eclipse Plus C₁₈柱(100 mm×2.1 mm, 1.8 μm)上实现3种贝类毒素的基线分离。该方法采用多反应监测(MRM)模式扫描,采用标准曲线外标法定量。3种原多甲藻酸在1~100 μg/kg范围内线性关系良好,相关系数(r²)均大于0.995; 3种贝类毒素的定量限(S/N=10)均为1.0 μg/kg;在10、20和50 μg/kg 3个加标水平的添加回收率在71%~108%之间,日内和日间测定的相对标准偏差≤10%(n=6)。应用该方法对国内外多个地区的贝类产品进行了筛查测定,发现部分样品的测定结果为阳性。该方法灵敏度高,重复性好,操作简便、快捷,适用于食用贝类及其制品中3种原多甲藻酸贝类毒素的分析测定。     

准静态扫集胶束电动毛细管色谱法测定扇贝样品中的贝类毒素

采用准静态扫集胶束电动毛细管色谱(MEKC)法测定了扇贝样品中的2种贝类毒素。毛细管内首先充满含十二烷基硫酸钠(SDS)的缓冲溶液,调节缓冲溶液的pH值,使电渗流等于SDS胶束的电泳流速,电动进样时,带正电荷的贝类毒素离子被SDS扫集吸附,由于SDS在毛细管内处于准静止状态,可使进样时间延长至320 s。与常规电动进样MEKC相比,石房蛤毒素和软骨藻酸的检测灵敏度分别提高950和810倍。该方法对石房蛤毒素和软骨藻酸的检出限分别为0.05,0.12 ng/m L。方法可实现对扇贝样品中2种贝类毒素的快速、灵敏检测。     

反相高效液相色谱法测定3种中成药中的葛根素

 建立了测定小儿清感灵片、步长新脑心通胶囊和感冒清热颗粒 3种中成药中葛根素含量的反相高效液相色谱方法。采用APEXODS色谱柱 ,以醋酸铵缓冲液 (10 0mmol/L ,pH 5 0 ) 甲醇 (体积比为 75∶2 5 )的混合溶液为流动相 ,检测波长为 2 5 0nm ,流速为 0 8mL/min。葛根素在 2mg/L～ 2 0mg/L时其色谱峰面积与质量浓度的线性关系良好 (r =0 9999) ;上述 3种中成药中的葛根素含量分别为 3 48mg/g ,1 0 8mg/ g及 1 5 2mg/ g(蔗糖型 ) ;其加样回收率分别为 99 0 % ,93 4%和 97 5 %。该法简便、快速、专属性强 ,可以作为多种中药制剂中葛根素含量的测定方法。     

高效液相色谱法快速测定抗真菌制剂M18中的吩嗪-1-羧酸

 建立了测定抗真菌制剂M18中吩嗪 1 羧酸的反相高效液相色谱方法。流动相为甲醇 5mmol/L磷酸缓冲液(pH 5 0 ) (体积比为 6 0∶40 ) ,流速为 1mL/min ,检测波长为 2 48nm ,线性范围是 5 0mg/L～ 5 0 0mg/L ,检测限是 30mg/L ,回收率为 97 5 3% ,RSD为 1 5 %。该方法具有快速、简便、灵敏、重现性好的特点。     

液相色谱-串联质谱法测定虾夷扇贝和长牡蛎中软骨藻酸的残留

建立了液相色谱-串联质谱测定虾夷扇贝和长牡蛎中贝类毒素软骨藻酸残留的检测方法。样品经50%甲醇提取,LC-SAX柱净化,3mL0.1mol/L甲酸溶液洗脱,电喷雾离子源(ESI),在正离子、多反应监测方式(MRM)模式下进行定性与定量,定性离子对为m/z 311.98/265.91,m/z 311.98/247.9,m/z 311.98/192.91,以m/z 311.98/265.91为定量离子对,外标法定量。结果表明,方法的检测限为0.01μg/g,定量限为0.02μg/g。在0.02~10μg/mL范围内线性相关系数为0.9999。当添加软骨藻酸质量分数为20~1000 ng/g时,虾夷扇贝样品中软骨藻酸的平均回收率为81.3%~105.4%,RSD为3.9%~8.9%(n=6);长牡蛎样品中软骨藻酸的平均回收率为83.5%~106.6%,RSD为4.6%~6.4%(n=6)。方法满足对贝类产品中软骨藻酸残留的测定。     

液相色谱-串联质谱检测贝类组织中5种脂溶性贝毒素

建立了液相色谱-串联质谱分析贝类组织中米氏裸甲藻(GYM)贝毒素、螺环内酯毒素(SPX1)、大田软骨酸(OA)贝毒素、蛤毒素(PTX2)、原多甲藻酸(AZA1)贝毒素的方法.用甲醇-水(4: 1, V/V)溶液对贝类组织中GYM, SPX1, OA, PTX2和AZA1进行提取,MAX阴离子交换柱净化后,采用液相色谱分离,除OA以负离子选择反应监测外,GYM, SPX1, PTX2和AZA1以电喷雾离子源正离子选择反应监测模式进行质谱分析.5种脂溶性贝毒素GYM, SPX1, OA, PTX2和AZA1在各自相应浓度范围内线性良好,相关系数＞0.99.扇贝闭壳肌空白样品添加5种贝毒素的提取率均为78.6%～94.4%(n=6); 精密度(RSD)为6.8%～14.9%.贝类组织中5种贝毒素GYM, SPX1, OA, PTX2和AZA1的检出限分别为0.10, 0.21, 2.00, 0.32和0.04 μg/kg.     

高效液相法测定注射用赖氨匹林中的阿司匹林及游离水杨酸

 建立了高效液相法 (HPLC)同时测定注射用赖氨匹林中阿司匹林和游离水杨酸含量的方法。采用的柱为HypersilBDSC18柱 ,流动相为甲醇 水 冰醋酸 (体积比为 35∶6 5∶3) ,检测波长为 2 80nm。阿司匹林和水杨酸的质量浓度分别为 0 0 2 8g/L～ 0 14 1g/L和 0 77mg/L～ 3 85mg/L时线性关系良好 ,其线性相关系数分别为0 9999和 0 9998;加样回收率分别为 99 2 7% (RSD =0 8% )及 99 6 1% (RSD =1 3% )。     

快速高分离度液相色谱法测定贝类中软骨藻酸残留量

介绍了用快速高分离度液相色谱(RRIC)测定贝类中记忆丧失性贝类毒素软骨藻酸的方法.样品以V(甲醇):V(水)=1:1的溶液提取,经LC-SAX强阴离子柱固相萃取净化,以ZORBAX SB-C18柱(1.8μm,2.1×50mm)分离,乙腈+0.1%三氟乙酸水溶液等度洗脱,1 min内完成检测.用RRIC外标法定量分析.在10,15,20,40 mg/kg 4个添加水平下进行了验证试验,方法在0～18mg/L范围内有良好的线性关系,相关系数大于0.999,回收率为79.3%～106%.     

液相色谱-串联质谱法检测贝类产品中的原多甲藻酸贝类毒素

建立了一种贝类组织中原多甲藻酸(azaspiracid, AZA)贝类毒素主要成分AZA1的高效液相色谱-串联质谱检测方法。本方法采用甲醇-水(80:20, v/v)溶液对贝类组织中AZA1进行提取,并用MAX阴离子交换固相萃取(SPE)柱富集净化,使用Atlantis dC18(150 mm×4.6 mm, 5.0 μm)色谱柱分离,以含有50 mmol/L甲酸和2 mmol/L甲酸铵的乙腈-水溶液(80:20, v/v)为流动相进行等度洗脱,质谱采用选择反应监测(SRM)模式。AZA1在5 min内获得完全分离,且在48.85～2 442 ng/L范围内线性良好,相关系数为0.998 1。该方法检出限(S/N=3)为11.00 pg/g,添加水平为36.64、73.27、146.54 pg/g时的平均回收率为75.8%～82.5%(n=6),相对标准偏差小于10%。利用该方法对采自大连、青岛、广州水产品市场上的112个贝类样品进行了分析,发现采自大连和广州的部分贝类样品中含有AZA1。结果表明,该方法具有简单、快速、灵敏度高等特点,能充分满足贝类中AZA1检测的要求。     

Determination of domoic acid in seawater and phytoplankton by liquid chromatography-tandem mass spectrometry

Domoic acid (DA) is an algal neurotoxin produced by diatoms primarily of the genus Pseudo-nitzschia and is responsible for the human intoxication syndrome known as amnesic shellfish poisoning. A method has been developed to determine DA in seawater and phytoplankton matrices by liquid chromatography-tandem mass spectrometry for both quantitation and confirmation purposes. Sample extraction and clean-up was achieved on a C18 solid-phase extraction (SPE) cartridge. An acidic condition is critical for retaining hydrophilic DA on the cartridge. Direct injection of SPE eluate for analysis is recommended in order to avoid loss of DA by drying with heat prior to resuspension and injection. DA was quantified using the fragments produced from the protonated DA ion through multiple reaction monitoring (MRM). Recoveries exceeded 90% for all seawater samples spiked with DA and approximated 98% of toxin standard added to cultured phytoplankton material. Acceptable reproducibility (ca. 5% or less) was obtained for all intra-day and inter-day samples. The detection limit was 30 pg/ml level with a 20 microl injection volume, which demonstrated the value of this method for not only confirming DA production by minimally toxic phytoplankton species, but also for investigating the potentially important role of dissolved DA in marine food webs.     

New fluorimetric method of liquid chromatography for the determination of the neurotoxin domoic acid in seafood and marine phytoplankton

Domoic acid (DA) is a neurotoxic amino acid that is responsible for the human toxic syndrome, amnesic shellfish poisoning (ASP). A new rapid, sensitive liquid chromatographic (LC) method has been developed for the determination of DA in various marine samples. DA in marine biological materials was derivatised with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and analysed using isocratic reversed-phase LC with fluorimetric detection. The calibration, based on standard DA solutions, was linear in the range 0.04-2 microg/ml (r2=0.998) and the detection limit (3:1, signal/noise) was better than 1 ng/ml. Using the certified reference material (MUS-1B), recoveries of DA from shellfish tissue were >95% (n=5). When a strong anion exchange SPE cartridge was used for sample clean-up the detection limit was 6 ng DA/g mussel tissue. Good reproducibility was achieved with RSD values ranging from 3% for 8 microg DA/g (n=5), to 5% for 0.04 microg DA/g (n=5). This new method was successfully applied to the determination of DA in naturally contaminated shellfish and in marine phytoplankton cultures of Pseudonitzschia sp.     

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.     

Evolving to the optoelectronic mouse for phycotoxin analysis in shellfish

Despite ethical and technical concerns, the in vivo method, or more commonly referred to mouse bioassay (MBA), is employed globally as a reference method for phycotoxin analysis in shellfish. This is particularly the case for paralytic shellfish poisoning (PSP) and emerging toxin monitoring. A high-performance liquid chromatography method (HPLC-FLD) has been developed for PSP toxin analysis, but due to difficulties and limitations in the method, this procedure has not been fully implemented as a replacement. Detection of the diarrhetic shellfish poisoning (DSP) toxins has moved towards LC-mass spectrometry (MS) analysis, whereas the analysis of the amnesic shellfish poisoning (ASP) toxin domoic acid is performed by HPLC. Although alternative methods of detection to the MBA have been described, each procedure is specific for a particular toxin and its analogues, with each group of toxins requiring separate analysis utilising different extraction procedures and analytical equipment. In addition, consideration towards the detection of unregulated and emerging toxins on the replacement of the MBA must be given. The ideal scenario for the monitoring of phycotoxins in shellfish and seafood would be to evolve to multiple toxin detection on a single bioanalytical sensing platform, i.e. ‘an artificial mouse’. Immunologically based techniques and in particular surface plasmon resonance technology have been shown as a highly promising bioanalytical tool offering rapid, real-time detection requiring minimal quantities of toxin standards. A Biacore Q and a prototype multiplex SPR biosensor have been evaluated for their ability to be fit for purpose for the simultaneous detection of key regulated phycotoxin groups and the emerging toxin palytoxin. Deemed more applicable due to the separate flow channels, the prototype performance for domoic acid, okadaic acid, saxitoxin, and palytoxin calibration curves in shellfish achieved detection limits (IC₂₀) of 4,000, 36, 144 and 46 μg/kg of mussel, respectively. A one-step extraction procedure demonstrated recoveries greater than 80 % for all toxins. For validation of the method at the 95 % confidence limit, the decision limits (CCα) determined from an extracted matrix curve were calculated to be 450, 36 and 24 μg/kg, and the detection capability (CCβ) as a screening method is ≤10 mg/kg, ≤160 μg/kg and ≤400 μg/kg for domoic acid, okadaic acid and saxitoxin, respectively.     

Determination of submicrogram-per-liter concentrations of caffeine in surface water and groundwater samples by solid-phase extraction and liquid chromatography

A method for determining submicrogram-per-liter concentrations of caffeine in surface water and groundwater samples has been developed. Caffeine is extracted from a 1 L water sample with a 0.5 g graphitized carbon-based solid-phase cartridge, eluted with methylene chloride-methanol (80 + 20, v/v), and analyzed by liquid chromatography with photodiode-array detection. The single-operator method detection limit for organic-free water samples was 0.02 microgram/L. Mean recoveries and relative standard deviations were 93 +/- 13% for organic-free water samples fortified at 0.04 microgram/L and 84 +/- 4% for laboratory reagent spikes fortified at 0.5 microgram/L. Environmental concentrations of caffeine ranged from 0.003 to 1.44 micrograms/L in surface water samples and from 0.01 to 0.08 microgram/L in groundwater samples.     

Single-laboratory validation of the biosense direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of domoic acid toxins in shellfish

Method validation was conducted for an enzyme-linked immunosorbent assay (ELISA) for the determination of domoic acid (DA) toxins, known to give amnesic shellfish poisoning (ASP) symptoms, in shellfish. The calibration curve range of the assay is approximately 10-260 pg/mL, with a dynamic working range for DA toxins in shellfish from 0.01 to at least 250 mg/kg. The ASP ELISA showed no significant cross-reactivity to structural analogs, and proved to be robust to deliberate alterations of the optimal running conditions. The shellfish matrix effects observed with mussels, oysters, and scallops were eliminated by diluting shellfish extracts 1:200 prior to analysis, leading to a limit of detection at 0.003 mg/kg. Thirteen blank shellfish homogenates were spiked with certified mussel material containing DA to levels in the range of 0.1-25 mg DA/kg, and analyzed in quadruplicate on 3 different days. The relative standard deviation (RSD) under intra-assay repeatability conditions ranged from 6.5 to 13.1%, and under interassay repeatability conditions the RSD ranged from 5.7 to 13.4%, with a mean value of 9.3%. The recoveries ranged from 85.5 to 106.6%, with a mean recovery of 102.2%. A method comparison was conducted with liquid chromatography with ultraviolet detection, using naturally contaminated scallop samples (n = 27) with DA levels at 0-244 mg/kg. The overall correlation coefficient was 0.960 and the slope of the regression was 1.218, indicating a good agreement between the methods.     

Determination and confirmation of the amnesic shellfish poisoning toxin, domoic acid, in shellfish from Scotland by liquid chromatography and mass spectrometry

During 1998 and early 1999, shellfish samples from sites in Scotland were found to contain the amnesic shellfish poisoning toxin, domoic acid (DA). Two different techniques, liquid chromatography (LC) with UV diode-array detection and LC with mass spectrometric (MS) detection, were used to detect and confirm DA in shellfish extracts. The LC/UV method was validated for routine monitoring by recovery experiments on spiked mussel and scallop tissues with a certified mussel tissue used as reference material. Crude extracts of selected samples as well as extracts cleaned with strong anion exchange (SAX) were analyzed by both LC/UV and LC/MS. Good correlation (linear regression r2 = 0.996, slope = 0.93) between the 2 methods was found for cleaned extracts. Analyses of crude extracts by LC/UV produced false-positive results in 2 crab samples, whereas LC/MS analyses gave accurate results. It was concluded that LC/UV is a valid approach for routine monitoring of DA in shellfish when cleanup is performed with a SAX cartridge to prevent false positives. A variety of shellfish species were surveyed for DA content, including Pecten maximus (king scallops), Chlamys opercularis (queen scallop), Mytilus edulis (blue mussels), Cancer pugaris (crab), and Ensis ensis (razor fish). The highest concentration of DA was 105 microg/g in Pecten maximus.     

Determination of domoic acid in shellfish extracted by molecularly imprinted polymers

A selective sample cleanup method using molecularly imprinted polymers was developed for the separation of domoic acid (a shellfish toxin) from shellfish samples. The molecularly imprinted polymers for domoic acid was prepared by emulsion polymerization using 1,3,5‐pentanetricarboxylic acid as the template molecule, 4‐vinyl pyridine as the functional monomer, ethylene glycol dimethacrylate as the crosslinker, and Span80/Tween‐80 (1:1 v/v) as the composite emulsifiers. The molecularly imprinted polymer showed high affinity to domoic acid with a dissociation constant of 13.5 μg/mL and apparent maximum adsorption capacity of 1249 μg/g. They were used as a selective sorbent for the detection of domoic acid from seafood samples coupled with high‐performance liquid chromatography. The detection limit of 0.17 μg/g was lower than the maximum level permitted by several authorities. The mean recoveries of domoic acid from clam samples were 93.0–98.7%. It was demonstrated that the proposed method could be applied to the determination of domoic acid from shellfish samples.     

Confirmation of domoic acid in shellfish using butyl isothiocyanate and reversed-phase liquid chromatography.

A simple chemical confirmatory technique has been developed for domoic acid, a neurotoxic amino acid of marine origin. After extraction with water-methanol, the domoic acid-containing extract is analysed directly by reversed-phase liquid chromatography with UV absorption detection at 242 nm. For confirmation of positive results an aliquot of the extract is evaporated to dryness and reacted with butyl isothiocyanate to form a thiourea derivative which elutes later than underivatized domoic acid. No additional sample cleanup is required in order to carry out the derivatization for conformation of domoic acid at the Canadian 20 micrograms/g guideline level in shellfish. In mussel extract, domoic acid was converted to the thiourea derivative with a yield of 86-91% compared to a pure standard carried through the same reaction. The detection limit for the derivative was about 5-10 micrograms/g of equivalent domoic acid in extracts of mussels, clams or oysters.