Screening of antibiotics in surface and wastewater samples by ultra-high-pressure liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry

The potential of ultra-high-pressure liquid chromatography (UHPLC) coupled to hybrid quadrupole time-of-flight mass spectrometry (QTOF–MS) for the screening and confirmation of antibiotics in water samples is illustrated in this paper. UHPLC presents several advantages over conventional liquid chromatography as it generates narrow peaks (increasing peak height and improving sensitivity) and reduces chromatographic runs. Regarding QTOF–MS, its increased mass resolution, high sensitivity in full-spectrum acquisition mode and mass accuracy, in both MS and MS/MS modes, make this technique ideal for the detection and reliable confirmation of organic contaminants in environmental samples. After a solid-phase extraction using Oasis HLB cartridges, UHPLC–QTOF–MS has been applied in this work to several types of water samples (surface water and influent and effluent wastewaters). Several antibiotics were found in the samples, such as ofloxacin, ciprofloxacin, clarythromycin or erythromycin, among others. Moreover, the full spectrum data provided by TOF–MS acquisition has enabled searching for many other pharmaceuticals that could be present in the samples in a “post-target” way. This approach has allowed the detection and confirmation of paracetamol, omeprazole and codeine, among others. UPLC–QTOF–MS has been shown as an attractive and efficient hyphenated technique for the rapid detection and confirmation of pharmaceuticals in water with very little sample handling.     

Glossary of terms for separations coupled to mass spectrometry

This document is a glossary of terms for separations coupled to mass spectrometry. It covers gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry, and supercritical fluid chromatography/mass spectrometry and the sample introduction, ionization, and data analysis methods used with these combined techniques.     

Liquid chromatography with time-of-flight mass spectrometry for simultaneous determination of chemotherapeutant residues in salmon

Liquid chromatography with time-of-flight mass spectrometry (LC-TOF-MS) method has been developed for simultaneous confirmation by accurate mass measurement and quantitative determination of antibiotics (enrofloxacin, oxolinic acid, flumequine, erythromycin), fungicides (malachite green MG, leucomalachite green LMG) and parasiticide (emamectin benzoate) residues in edible portion of salmon. Confirmation of chemotherapeutant residues has been based on the system of identification points (IPs) established in the Commission Decision 2002/657/EC concerning the use of mass spectrometry (MS) techniques. A validation study on matrix is presented evaluating accuracy in terms of precision (λ_ppm 0.83-1.15) and trueness (0.22-0.70 Da). Limits of detection (LODs) and limits of quantification (LOQs) were in ranges of 1-3 and 3-9 μg/kg, below the maximum residue limits (MRLs) established in current EU legislation (100-200 μg/kg) for these chemotherapeutants. Considering the EU guidelines, decision limits (CCα) and detection capabilities (CCβ) were determined (ranges of 103-218 and 107-234 μg/kg, respectively) for authorised substances. For no authorised compounds (MG and LMG), LODs were 2 and 1 μg/kg, respectively, but exceed the MRPL (minimum required performance limit) established in the legislation which corresponds to the sum of MG and LMG (2 μg/kg). Acceptable intra-day and inter-day variability, in terms of relative standard deviation (R.S.D.) of the analytical method, were obtained (2-15%). Linearity was demonstrated from the LOQs of the analytes to 600 μg/kg (r > 0.9991). The method has involved an extraction procedure based on solid-liquid extraction (SLE) with recoveries higher than 80% for most target chemotherapeutants, with exception of enrofloxacin (40%).     

Determination of 19-nortestosterone residues in aquaculture tissues by enzyme-linked immunosorbent assay and comparison with liquid chromatography and tandem mass spectrometry

19-Nortestosterone (17β-NT) was oximated by carboxymethoxylamine and then coupled with bovine serum albumin (BSA) in a mixed-anhydride reaction in order to produce an antibody. The conjugate rate of 17β-NT and BSA was estimated to be 24 by ultraviolet spectrophotometry. Polyclonal antibody of 17β-NT was acquired from the animal immunized with the conjugate. Through an indirect enzyme-linked immunosorbent assay (ELISA), which demonstrated that the synthesis of immunogen was successful, the titre of antiserum was found to be 6.4?×?10⁵. Based on the purified antibody, a competitive indirect ELISA was developed. ELISA revealed that the limit of detection (LOD) was 0.07?ng?g^?1, the recovery (in edible tissues) was 71–89%, and the working range was 0.05–31.25?ng?g^?1. The preliminary evaluation of assay performance through specificity, sensitivity, precision, and accuracy revealed that this ELISA method could be used in the practical detection of 17β-NT in tissue samples. Moreover, this method was compared with high-performance liquid chromatography tandem mass spectrometry, for which the transition for quantification of 17β-NT was 275.4/109.1.     

Analysis of keto-enol tautomers of curcumin by liquid chromatography/mass spectrometry

Keto-enol tautomers of curcumin were confirmed by reversed-phase liquid chromatography(RPLC)/ hybrid quadrupole ion trap/time-of-flight mass spectrometry(QIT/TOFMS).Tautomers gave different MS/MS spectra in negative mode.Different mass spectra were also obtained by hydrogen/deuterium exchange LC/MS/MS in positive mode.Our results suggest that enol form is the major form in the solution(water/acetonitrile).     

Combination of liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry for the detection of 21 anabolic steroid residues in bovine urine

For the detection of anabolic steroid residues in bovine urine, a highly sensitive liquid chromatographic/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method was developed using both positive and negative ionization. For four compounds the ESI mode was not sensitive enough and gas chromatographic/mass spectrometric GC/MS detection was therefore still necessary as a complementary method. The sample clean-up consisted of solid-phase extraction (SPE) on a C(18) column followed by enzymatic hydrolysis and a second solid-phase extraction on a combination of a C(18) and a NH(2) column. After this last SPE clean-up, the eluate was split into two equal aliquots. One aliquot was further purified and after derivatization used for GC/MS analysis. The other aliquot was analyzed with LC/MS/MS in both ESI+ and ESI- modes. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) were between 0.16 and 1 ng ml(-1) for the compounds detected with the LC/MS/MS method. The developed method is used in routine analysis in our laboratory.     

Solid-phase extraction followed by liquid chromatography quadrupole time-of-flight tandem mass spectrometry for the selective determination of fungicides in wine samples

In this work, a reliable and selective procedure for the determination of thirteen fungicides in red and white wine samples is proposed. Solid-phase extraction (SPE) and liquid chromatography (LC) tandem mass spectrometry (MS/MS), based on a hybrid quadrupole time-of-flight (QTOF) system, were used as sample preparation and determination techniques, respectively. Extraction and purification of target analytes was carried out simultaneously by using a reversed-phase Oasis HLB (200mg) SPE cartridge combined with acetonitrile as elution solvent. Fungicides were determined operating the electrospray source in the positive ionization mode, with MS/MS conditions adjusted to obtain at least two intense product ions per compound, or registering two transitions per species when a single product was noticed. High selective MS/MS chromatograms were extracted using a mass window of 20 ppms for each product ion. Considering external calibration as quantification technique, the overall recoveries (accuracy) of the procedure ranged between 81% and 114% for red and white wine samples (10-20 mL), spiked at different concentrations between 5 and 100 ng mL(-1). Relative standard deviations of the above data stayed below 12% and the limits of quantification (LOQs) of the method, calculated for 10 mL of wine, varied between 0.1 ng mL(-1) for cyprodinil (CYP) and 0.7 ng mL(-1) for myclobutanil (MYC). The optimized method was applied to seventeen commercial wines produced in Spain and obtained from local supermarkets. Nine fungicides were determined, at levels above the LOQs of the method, in the above samples. The maximum concentrations and the highest occurrence frequencies corresponded to metalaxyl (MET) and iprovalicarb (IPR).     

Determination of antimicrobial residues and metabolites in the aquatic environment by liquid chromatography tandem mass spectrometry

Antimicrobials are used in large quantities in human and veterinary medicine. Their environmental occurrence is of particular concern due to the potential spread and maintenance of bacterial resistance. After intake by the organisms, the unchanged drug and its metabolized forms are excreted and enter wastewater treatment plants where they are mostly incompletely eliminated, and are therefore eventually released into the aquatic environment. The reliable detection of several antimicrobials in different environmental aqueous compartments is the result of great improvements achieved in analytical chemistry. This article provides an overview of the more outstanding analytical methods based on liquid chromatography tandem mass spectrometry, developed and applied to determine antimicrobial residues and metabolites present in surface, waste, and ground waters.      

高效液相色谱-串联质谱法测定肠衣中17种磺胺类药物残留量

建立了一种以13C6-磺胺二甲基嘧啶为内标快速测定肠衣中17种磺胺类药物残留量的方法.样品经过V(乙腈)∶V(乙酸乙酯)=1∶1 混合溶剂提取后,离心,上清液在旋转蒸发仪上蒸发至干,体积分数30%甲醇溶液定容,正己烷除去脂肪,经过滤膜后,进高效液相色谱-串联四极杆质谱进行分析,内标法定量.方法的回收率在80%～100%之间,相对标准偏差在15.4%以下,定量限为1.0 μg/kg(磺胺脒为2.0 μg/kg),检出限在0.4 μg/kg以下(磺胺脒为1.0 μg/kg).方法适合于大批量样品的快速检测.     

凝胶渗透色谱-气相色谱串联质谱法测定动物脂肪中164种农药残留

建立了气相色谱-串联质谱法(GC-MS-MS)同时测定动物脂肪中164种农药的多残留分析方法。样品经乙腈均质提取2次,合并提取液并在45℃水浴中旋转浓缩至1 mL,加入5 mLV(环己烷)∶V(乙酸乙酯)=1∶1进行溶剂交换2次后,用V(环己烷)∶V(乙酸乙酯)=1∶1稀释至10 mL,用凝胶渗透色谱(GPC)净化,GC-MS-MS多反应监测模式(MRM)测定,内标法定量。本方法研究的164种农药的线性相关系数为0.9438~1.0000,其中有154种在0.9900以上,占总数的93.9%。方法检出限(S/N≥3)为0.1~360.0μg/kg,其中在10.0μg/kg以下的有121种,占研究农药总数的73.8%。用猪、鸡、牛、羊4种不同的脂肪在低、中、高3个添加水平评价了164种农药的方法效率,其中有150种的回收率在70%~120%之间,相对标准偏差(RSDs)在20%以内。本方法适用于不同动物脂肪中150种农药多残留的定量测定和14种农药的定性测定。     

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.     

Evaluation of analytical instrumentation. Part XXII Instrumentation for liquid chromatography/mass spectrometry

The reports of this series tabulate a number of features of analytical instruments that should be considered when making comparison between various systems. Scoring these features in a rational manner allows a scientific comparison to be made between instruments as an aid to selection. This is the XXIInd report of the series and deals with instrumentation for liquid chromatography/mass spectrometry.     

Determination of benzophenones in human placental tissue samples by liquid chromatography-tandem mass spectrometry

Benzophenones (BPs) are a family of compounds widely used to protect the skin and hair from UV irradiation. Despite human exposure to BPs through dermal application of products containing sunscreen agents and the increasing evidence that BPs are able to interfere with endocrine systems, few studies have examined the occurrence of BPs in humans. In this work, we propose a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine six BPs, namely, benzophenone-1 (BP-1), benzophenone-2 (BP-2), benzophenone-3 (BP-3), benzophenone-6 (BP-6), benzophenone-8 (BP-8) and 4-hydroxybenzophenone (4-OH-BP) in human placental tissue samples. The method involves an extraction step of the analytes from the samples using ethyl acetate, followed by a clean-up step using centrifugation prior to their quantification by LC-MS/MS using an atmospheric pressure chemical ionization (APCI) interface in the positive mode. Benzophenone-d₁₀ (BP-d₁₀) was used as surrogate. Found detection limits (LOD) ranged from 0.07 to 0.3 ng g⁻¹ and quantification limits (LOQ) from 0.3 to 1.0 ng g⁻¹, while inter- and intra-day variability was under 5%. The method was validated using standard addition calibration and a recovery assay. Recovery rates for spiked samples ranged from 98 to 104%. This method was satisfactorily applied for the determination of BPs in 16 placental tissue samples collected from women who live in Granada (Spain).     

Determination of neonicotinoid pesticides residues in agricultural samples by solid-phase extraction combined with liquid chromatography-tandem mass spectrometry

This work reports a new sensitive multi-residue liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection, confirmation and quantification of six neonicotinoid pesticides (dinotefuran, thiamethoxam, clothiandin, imidacloprid, acetamiprid and thiacloprid) in agricultural samples (chestnut, shallot, ginger and tea). Activated carbon and HLB solid-phase extraction cartridges were used for cleaning up the extracts. Analysis is performed by LC-MS/MS operated in the multiple reaction monitoring (MRM) mode, acquiring two specific precursor-product ion transitions per target compound. Quantification was carried by the internal standard method with D(4)-labeled imidacloprid. The method showed excellent linearity (R(2)≥0.9991) and precision (relative standard deviation, RSD≤8.6%) for all compounds. Limits of quantification (LOQs) were 0.01 mg kg(-1) for chestnut, shallot, ginger sample and 0.02 mg kg(-1) for tea sample. The average recoveries, measured at three concentrations levels (0.01 mg kg(-1), 0.02 mg kg(-1) and 0.1 mg kg(-1) for chestnut, shallot, ginger sample, 0.02 mg kg(-1), 0.04 mg kg(-1) and 0.2 mg kg(-1) for tea sample), were in the range 82.1-108.5%. The method was satisfactorily validated for the analysis of 150 agricultural samples (chestnut, shallot, ginger and tea). Imidacloprid and acetamiprid were detected at concentration levels ranging from 0.05 to 3.6 mg kg(-1).     

Validation of a method for the analysis of quinolones residues in bovine muscle by liquid chromatography with electrospray ionisation tandem mass spectrometry detection

A liquid chromatography-tandem mass spectrometry method for the determination and confirmation of nine quinolones was optimised and validated according to Commission Decision 2002/657/EC. Analytes were extracted from veal muscle with water and extracts purified with 96-well plates Oasis HLB cartridges. Separation was carried out in a silica-based C₁₈ column (50 mm × 2.1 mm) with mobile phases consisting of water/acetonitrile mixtures containing acetic acid. Linear calibration curves in the ranges 4-400 and 50-800 ng g⁻¹, with correlation coefficients at least 0.995, were obtained for all the analytes. At concentration levels above 10 ng g⁻¹, quantification errors were lower than 10% and repeatability and within-laboratory reproducibility standard deviations below 6% and 10%, respectively. Decision limits and detection capabilities are reported.     

高效液相色谱-串联质谱法测定动物源食品中粘杆菌素、多粘菌素B的残留量

建立了动物源食品中粘杆菌素和多粘菌素B残留的高效液相色谱-串联质谱(HPLC-MS/MS)测定方法。样品用V(10%三氯乙酸水溶液):V(乙腈)=30:70提取,Oasis WCX SPE柱净化,LC-MS/MS电喷雾正离子多反应监测模式(ESI+-MRM)检测。分析物在0～250μg/kg的浓度范围内呈良好线性,线性相关系数>0.995。方法的定量限为10μg/kg。方法在三个添加水平的平均回收率在71.6%～78.9%之间,相对标准偏差在6.2%～12%之间。方法适用于动物源食品中粘杆菌素和多粘菌素B的定量及确证检测。     

Detection of singly- and doubly-charged quaternary ammonium drugs in equine urine by liquid chromatography/tandem mass spectrometry

Quaternary ammonium drugs (QADs) are anticholinergic agents some of which are known to have been abused or misused in equine sports. A recent review of literature shows that the screening methods reported thus far for QADs mainly cover singly-charged QADs. Doubly-charged QADs are extremely polar substances which are difficult to be extracted and poorly retained on reversed-phase columns. It would be ideal if a comprehensive method can be developed which can detect both singly- and doubly-charged QADs. This paper describes an efficient liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous detection and confirmation of 38 singly- and doubly-charged QADs at sub-parts-per-billion (ppb) to low-ppb levels in equine urine after solid-phase extraction.     

QuEChERS-高效液相色谱-串联质谱法测定动物源性食品中3种镇静剂残留

建立了QuEChERS-高效液相色谱-串联质谱(HPLC-MS/MS)同时测定猪肉、鱼肉、肝脏和肾脏中氯丙嗪、地西泮和安眠酮残留量的分析方法。样品用无水Na_2SO_4脱水、乙酸乙酯提取和C_(18)、N-丙基乙二胺(PSA)和氨基填料(NH2填料)净化,使用一种特殊的C_(18)色谱柱Atlantis T3,以5 mmol/L甲酸溶液和乙腈溶液为流动相进行梯度洗脱。在正离子电离多反应监测(MRM)模式下,采用同位素内标法进行定量分析。3种镇静剂在0.2~5.0μg/L范围内线性关系良好。3种镇静剂在4类基质中3个水平(0.5、1和5μg/kg)下的加标回收率为92.5%~117.8%,相对标准偏差(RSD)为0.7%~11.6%(n=6)。该法高效快捷,灵敏度高,基质适应范围广,适用于大批量样品的快速分析。     

Residue determination of cyromazine and its metabolite melamine in chard samples by ion-pair liquid chromatography coupled to electrospray tandem mass spectrometry

A method has been developed for the sensitive and selective determination of cyromazine and its metabolite melamine in chard samples. Both compounds are small polar basic molecules, making their determination at residue levels complicated. The method involves an extraction procedure with phosphate buffer and methanol using high-speed blender, the addition of tridecafluoroheptanoic acid (TFHA) as ion-pair reagent and the injection of the five-fold diluted extract on liquid chromatography coupled to electrospray tandem mass spectrometry (LC–ESI–MS/MS). The method has been validated for chard samples, spiked at 0.05 and 0.5 mg kg⁻¹. Quantification was carried out by using matrix-matched standards calibration and recoveries were satisfactory, with mean values for cyromazine of 103% and 93%, and relative standard deviations lower than 7%. In the case of melamine, recoveries were 89% and 86%, with relative standard deviations lower than 13%. A limit of quantification of 0.05 mg kg⁻¹ was obtained for both compounds, with the limit of detection below 0.01 mg kg⁻¹. The method, with very little sample handling and good sensitivity, was applied to the rapid determination of low residue levels of these compounds in chards from field residue trials. All the quality controls included during the analysis were satisfactory with average recoveries of 92% and 78% for cyromazine and melamine, respectively.