Evaluation of two derivatization reagents for the determination by LC-MS/MS of ammonia in cigarette mainstream smoke

Ammonia in cigarette mainstream smoke was quantified by LC-MS/MS after derivatization. Two different reagents, fluorescamine and dansyl chloride, were investigated, but only the latter gave stable derivatives; therefore, it was considered the most appropriate choice. Smoke samples were collected on a Cambridge filter pad followed by an impinger containing a solution of hydrochloric acid. Ammonia was then derivatized with a 18.5 mM solution of dansyl chloride in acetonitrile at 70 °C for 30 min in a vial with the internal standard, (15)ND(4)Cl. The resulting derivative was analyzed by LC-MS/MS detection with an atmospheric pressure chemical ionization (APCI) interface in the positive ionization mode using multiple-reaction monitoring (MRM). Good linearity was obtained in the concentration range of 0.02-1.65 μg/mL (r(2) ≥ 0.999), and the limit of detection (LOD) was established at 0.006 μg/mL. This method has the advantage of being sensitive, efficient, and reliable and is not hindered by interferences from the sample matrix. It should thus be considered a reference method of choice for the determination of ammonia in smoke.     

Analysis of coffee for the presence of acrylamide by LC-MS/MS

A variety of popular instant, ground, and brewed coffees were analyzed using a modified liquid chromatography-tandem mass spectrometry (LC-MS/MS) method specifically developed for the determination of acrylamide in foods. Coffee test portions were spiked with 13C3-labeled acrylamide as an internal standard prior to their extraction and cleanup. Ground coffees (1 g) and instant coffees (0.5 g) were extracted by shaking with 9 mL of water for 20 min. Brewed coffee test portions (9 mL) were taken through the cleanup procedure without further dilution with extraction solvent. Coffee test portions were cleaned up by passing 1.5 mL first through an Oasis HLB (hydrophilic/lipophilic copolymer sorbent) solid phase extraction (SPE) cartridge and then a Bond Elut-Accucat (cation and anion exchange sorbent) SPE cartridge. The cleaned up extracts were analyzed by positive ion electrospray LC-MS/MS. The MS/MS data was used to detect, confirm, and quantitate acrylamide. The limit of quantitation of the method was 10 ng/g for ground and instant coffees and 1.0 ng/mL for brewed coffee. The levels of acrylamide ranged from 45 to 374 ng/g in unbrewed coffee grounds, from 172 to 539 ng/g in instant coffee crystals, and from 6 to 16 ng/mL in brewed coffee.     

Multiclass determination and confirmation of antibiotic residues in honey using LC-MS/MS

A multiclass method has been developed for the determination and confirmation in honey of tetracyclines (chlortetracycline, doxycycline, oxytetracycline, and tetracycline), fluoroquinolones (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, and sarafloxacin), macrolides (tylosin), lincosamides (lincomycin), aminoglycosides (streptomycin), sulfonamides (sulfathiazole), phenicols (chloramphenicol), and fumagillin residues using liquid chromatography tandem mass spectrometry (LC-MS/MS). Erythromycin (a macrolide) and monensin (an ionophore) can be detected and confirmed but not quantitated. Honey samples (approximately 2 g) are dissolved in 10 mL of water and centrifuged. An aliquot of the supernatant is used to determine streptomycin. The remaining supernatant is filtered through a fine-mesh nylon fabric and cleaned up by solid phase extraction. After solvent evaporation and sample reconstitution, 15 antibiotics are assayed by LC-MS/MS using electrospray ionization (ESI) in positive ion mode. Afterward, chloramphenicol is assayed using ESI in negative ion mode. The method has been validated at the low part per billion levels for most of the drugs with accuracies between 65 and 104% and coefficients of variation less than 17%. The evaluation of matrix effects caused by honey of different floral origin is presented.     

Evaluation of a modified QuEChERS extraction of multiple classes of pesticides from a rice paddy soil by LC-APCI-MS/MS

A new method for the determination of clomazone, fipronil, tebuconazole, propiconazole, and azoxystrobin in samples of rice paddy soil is presented. The extraction of the pesticides from soil samples was performed by using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. Some extraction conditions such as salt addition, sample acidification, use of buffer, and cleanup step were evaluated. The optimized method dealt with a single extraction of the compounds under study with acidified acetonitrile, followed by the addition of MgSO(4) and NaCl prior to the final determination by liquid chromatography-atmospheric chemical pressure ionization-tandem mass spectrometry. Validation studies were carried out in soil samples. Recoveries of the spiked samples ranged between 70.3 and 120% with relative standard deviation lower than 18.2%. The limits of quantification were between 10 and 50 μg kg(-1). The method was applied to the analysis of real samples of soils where rice is cultivated.     

Determination of grayanotoxins in biological samples by LC-MS/MS

A rapid LC-MS/MS method was developed for the quantitative determination of grayanotoxins I, II, and III in rumen contents, feces, and urine. The grayanotoxins were extracted from solid samples with methanol. The methanol extract was diluted with water and cleaned up using a reversed phase solid phase extraction column. HPLC separation was performed by reversed phase HPLC using a gradient of water and methanol containing 1% acetic acid. Determination was by positive ion electrospray ionization and ion trap tandem mass spectrometry. Grayanotoxin I quantitation was based on fragmentation of the sodium adduct ion at m/z 435 to a product ion at m/z 375. Grayanotoxins II and III were quantitated on the basis of fragmentation of the ion at m/z 335 to the product ion at m/z 299. The method detection limits were 0.2 microg/g in rumen contents and feces and 0.05 microg/g in urine. Fortifications at the detection limits and 10 times the detection limits of bovine rumen contents, caprine feces, and ovine urine were recovered in the range 80-114%. The diagnostic utility of the method was tested by analyzing samples submitted to the veterinary toxicology laboratory.     

Rapid sample preparation method for LC-MS/MS or GC-MS analysis of acrylamide in various food matrices

A fast and easy sample preparation procedure for analysis of acrylamide in various food matrices was developed and optimized. In its first step, deuterated acrylamide internal standard is added to 1 g of homogenized sample together with 5 mL of hexane, 10 mL of water, 10 mL of acetonitrile, 4 g of MgSO4, and 0.5 g of NaCl. Water facilitates the extraction of acrylamide; hexane serves for sample defatting; and the salt combination induces separation of water and acetonitrile layers and forces the majority of acrylamide into the acetonitrile layer. After vigorous shaking of the extraction mixture for 1 min and centrifugation, the upper hexane layer is discarded and a 1 mL aliquot of the acetonitrile extract is cleaned up by dispersive solid-phase extraction using 50 mg of primary secondary amine sorbent and 150 mg of anhydrous MgSO4. The final extract is analyzed either by liquid chromatography-tandem mass spectrometry or by gas chromatography-mass spectrometry (in positive chemical ionization mode) using the direct sample introduction technique for rugged large-volume injection.     

Rugged LC-MS/MS survey analysis for acrylamide in foods

The described liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection of acrylamide in food entails aqueous room temperature extraction, SPE cleanup, and analysis by LC-MS/MS. The method is applicable to a wide variety of foods. [(13)C(3)]acrylamide is the internal standard. The limit of quantitation is 10 ppb (microg/kg). Data were obtained in duplicate from >450 products representing >35 different food types. The variability in analyte levels in certain food types suggests that it may be possible to reduce acrylamide levels in those foods.     

Simultaneous determination of orysastrobin and its isomers in rice using HPLC-UV and LC-MS/MS

Orysastrobin is a new strobilurin-type fungicide to control leaf and panicle blast and sheath blight in rice. An analytical method was developed to determine the residues of orysastrobin and its two isomers, the main metabolite F001 and the major impurity F033, in hulled rice by the use of high-performance liquid chromatography with ultraviolet photometry (HPLC-UV) and liquid chromatography with tandem mass spectrometry (LC-MS/MS). All compounds were extracted with acetone from hulled rice samples. The extract was diluted with saline water, and an extraction step using dichloromethane/n-hexane partition was used to recover analytes from the aqueous phase. An n-hexane/acetonitrile partition and Florisil column chromatography were employed to further remove interfering coextractives prior to instrumental analysis. An octadecylsilyl column was successfully applied to identify orysastrobin and its isomers in sample extracts. Net recovery rates of orysastrobin, F001, and F033 from fortified samples ranged from 80.6 to 114.8% using HPLC-UV and LC-MS/MS. Relative standard deviations for the analytical methods were all <20%, and the quantification limits of the method were in the 0.002-0.02 mg/kg range. The proposed methods were reproducible and sufficiently accurate to evaluate the terminal residue of orysastrobin and its isomers in rice.     

UPLC-MS/MS同时测定土壤中19种植物激素方法的建立和验证

[目的]本文旨在建立土壤中多种植物激素的提取及超高效液相色谱串联质谱法,并用此方法同时测定土壤中茉莉酸、吲哚-3-乙酸、反式玉米素,玉米素核苷、异戊烯基腺嘌呤、吲哚-3-丁酸、N6-异戊烯基腺嘌呤、独脚金内酯、玉米素、二氢玉米素核苷、吲哚-3-丙酸、吲哚-3-乙酸甲酯、二氢玉米素、吲哚-3-羧酸、茉莉酸甲酯、油菜素内酯、脱落酸、赤霉素、水杨酸共19种植物激素含量。[方法]以根标土壤为试验材料,采用异丙醇水甲酸(80∶19∶1,V/V/V)作为提取剂,经过超声、离心后得到植物激素提取液,提取液于常温条件下真空浓缩至干,采用甲醇进行复溶,得到植物激素待测液。采用Waters ACQUITYUPLC■HSST3色谱柱对19种植物激素进行分离,以0.30 mmol/L甲酸铵水溶液(含0.01%甲酸)作为流动相A和0.30 mmol/L甲酸铵乙腈(含0.01%甲酸)作为流动相B进行梯度洗脱,流速为0.3 mL/min,柱温为30℃;采用超高效液相色谱串联质谱,多反应监测离子模式进行定性定量分析,其中,茉莉酸、吲哚-3-乙酸、反式玉米素、玉米素核苷、异戊烯基腺嘌呤、吲哚-3-丁酸、N6-异戊烯基腺嘌呤、独脚金内酯、玉米素、二氢玉米素核苷、吲哚-3-丙酸、吲哚-3-乙酸甲酯、二氢玉米素、吲哚-3-羧酸、茉莉酸甲酯、油菜素内酯采用正离子模式扫描,脱落酸、赤霉素、水杨酸采用负离子模式扫描;采用外标法测定植物激素回收率。[结果]在本试验浓度范围内,上述19种植物激素浓度与对应峰面积的相关系数(r)均大于0.99,检出限介于0.02~1.06 ng/g之间,19种植物激素的加标回收率为70.2%~117%,相对标准偏差介于0.20%~7.3%之间。采用优化后的实验方法测定土壤中的植物激素,结果检测出吲哚-3-乙酸、吲哚-3-羧酸、吲哚-3-乙酸甲酯、水杨酸和独角金内酯5种植物激素,含量为0.55~5.79ng/g。[结论]本方法前处理不需要过夜浸提,加入二氯甲烷后,超声离心浓缩复溶后可直接进样检测,大大缩短了样品的前处理时间,超高效液相色谱串联质谱法操作简单,选择性好,灵敏度高,可实现土壤样品中多种植物激素的同时检测,为土壤中植物激素的深入研究提供了一种有效的研究方法。     

Determination of strophanthidin in ingesta and plant material by LC-MS/MS

An LC-MS/MS method was developed for the semiquantitative determination of strophanthidin glycosides in ingesta from animals. Strophanthidin glycosides were simultaneously extracted and hydrolyzed to the strophanthidin aglycone using aqueous methanolic hydrochloric acid and the extracts cleaned up using solid-phase extraction. Extracts were analyzed using reverse-phase HPLC coupled with positive ion electrospray mass spectrometry. Characteristic product ion spectra were produced by fragmentation of the [M + H](+) precursor ion for each analyte. Quantitation was performed using the internal standard method with digitoxigenin serving as the internal standard. The method detection limit was calculated to be 0.075 microg/g, and the limit of quantitation was calculated to be 0.24 microg/g for strophanthidin in control rumen samples. This method was used in diagnostic investigations to confirm fatal strophanthidin glycoside poisonings in horses.     

Bovine colostrum: determination of naturally occurring steroid hormones by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

The aim of this study was to collect further data about levels of endogenous hormones in foodstuffs derived from animal production. Because of expected higher concentrations of especially estrogens in colostrum compared to other foodstuffs, our investigation focused on this matrix. For evaluation of endogenous steroid hormones in separated colostrum (fat and defatted fraction) and colostrum powder, the relevant free and conjugated estrogens (estradiol-17β, estradiol-17α, estrone, and estriol) androgens (androstendione, 19nor-androstendione, 19nor-testosterone-17β, 19nor-testosterone-17α, testosterone-17β, and testosterone-17α), and progesterone were determined by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Upmost determined concentrations were found in the fat fraction, with 25.56 and 7.59 μg/L for estrone and androstendione, respectively. In defatted milk and colostrum powder, conjugated estrogens dominated, whereas total (free and conjugated) estrone (5.51 μg/L; 15.0 μg/kg) exceeded estradiol-17α (2.66 μg/L; 7.5 μg/kg) and estradiol-17β (2.28 μg/L; 3.3 μg/kg). Neither 19nor-steroids nor estriol were detected in colostrum fractions or processed colostrum.     

QuEChERS结合高效液相色谱-串联质谱法测定土壤中11类47种药物残留

本文建立了一种QuEChERS结合高效液相色谱–串联质谱(UPLC-MS/MS)同时测定土壤中11类47种药物残留的分析方法。试验考察不同色谱柱和流动相对47种药物的分离效果,探究不同pH的EDTA-Mcllvaine缓冲液和净化填料对目标化合物的提取和净化效果,实现了对土壤中多类药物的定量分析。47种药物在对应线性范围内均呈现较好的线性关系(r≥0.995);方法的检出限为0.1 ~ 2.5 μg/kg,定量限为0.5 ~ 8 μg/kg;3水平加标试验的回收率为61.1% ~ 117.0%,相对标准偏差为1.9% ~ 11.4%。该方法适用于土壤多组分准确高通量定量分析,检出限、回收率、准确度均符合分析要求,为土壤中多种药物的赋存底数和潜在风险研究提供技术支撑。     

Mixed acid solubilization procedure for determination of total mercury in food samples

A mixed acid solubilization procedure has been developed for the determination of total mercury in food samples. This procedure has eliminated the problem of mercury loss from samples that are high in lipid or carbohydrate content. The solubilization is rapid and quantitative and can be used for a wide range of food items.     

High-throughput method for the quantitation of total folate in whole blood using LC-MS/MS

A high-throughput liquid chromatography tandem mass spectrometry (HT LC-MS/MS) method for red blood cell (RBC) folate analysis was developed from a previously described manual (M) LC-MS/MS method. The HT LC-MS/MS method used 96-well plates in which RBC folates were hydrolyzed with concentrated HCl in the presence of the [13C6]pABA internal standard (IS). The pH of the hydrolysate was adjusted to 5.0 before cleanup using 96-well plate OASIS HLB SPE cartridges. The analyte and IS were eluted with ethyl acetate/hexane (95:5, v/v) and methylated with methanol and trimethylsilyldiazomethane. The methylated analyte and IS were quantified with LC-MS/MS as previously described. The HT LC-MS/MS method was validated by determining the recovery of six different folate vitamers, which were quantitatively recovered (84-105% with CV<9.0%). RBC folate concentrations in whole blood samples correlated between HT and M LC-MS/MS methods (r=0.922, p<0.0001 for n=43 samples) and between the HT LC-MS/MS method and a chemiluminescence assay (r=0.664, p<0.001 for n=325 samples). Comparison of the results between HT LC-MS/MS and chemiluminescence methods with Bland-Altman difference plots and by ROC curve analysis indicates that the chemiluminescence assay underreports RBC folate concentrations. The HT LC-MS/MS method allows for high-throughput sample preparation for the analysis of RBC folate.     

Structure determination and sensory analysis of bitter-tasting 4-vinylcatechol oligomers and their identification in roasted coffee by means of LC-MS/MS

Aimed at elucidating intense bitter-tasting molecules in coffee, various bean ingredients were thermally treated in model experiments and evaluated for their potential to produce bitter compounds. As caffeic acid was found to generate intense bitterness reminiscent of the bitter taste of a strongly roasted espresso-type coffee, the reaction products formed were screened for bitter compounds by means of taste dilution analysis, and the most bitter tastants were isolated and purified. LC-MS/MS as well as 1-D/2-D NMR experiments enabled the identification of 10 bitter compounds with rather low recognition threshold concentrations ranging between 23 and 178 micromol/L. These bitter compounds are the previously unreported 1,3-bis(3',4'-dihydroxyphenyl) butane, trans-1,3-bis(3',4'-dihydroxyphenyl)-1-butene, and eight multiply hydroxylated phenylindanes, among which five derivatives are reported for the first time. In addition, the occurrence of each of these bitter compounds in a coffee brew was verified by means of LC-MS/MS (ESI-) operating in the multiple reaction monitoring (MRM) mode. The structures of these bitter compounds show strong evidence that they are generated by oligomerization of 4-vinylcatechol released from caffeic acid moieties upon roasting.     

Development and validation of an HPLC/UV/MS method for simultaneous determination of 18 preservatives in grapefruit seed extract

Grapefruit seed extracts are used in cosmetics, food supplements, and pesticides because of their antimicrobial properties, but suspicions about the true nature of the active compounds arose when synthetic disinfectants such as benzethonium or benzalkonium chloride were found in commercial products. The HPLC method presented herein allows the quality assessment (qualitative and quantitative) of these products for the first time. On the basis of a standard mixture of 18 preservatives most relevant for food and grapefruit products, a method was developed allowing the baseline separation of all compounds within 40 min. Optimum results were obtained with a C-8 stationary phase and a solvent system comprising aqueous trifluoroacetic acid, acetonitrile, and 2-propanol. The assay was fully validated and shown to be sensitive (LOD < or= 12.1 ng on-column), accurate (recovery rates > or = 96.1%), repeatable (sigma(rel) < or = 3.5%), precise (intra-day variation < or = 4.5%, interday variation < or = 4.1%), and rugged. Without any modifications the method could be adopted for LC-MS experiments, where the compounds of interest were directly assignable in positive ESI mode. The quantitative results of several products for ecofarming confirmed previous studies, as seven out of nine specimens were adulterated with preservatives in varying composition. The samples either contained benzethonium chloride (2.5-176.9 mg/mL) or benzalkonium chloride (138.2-236.3 mg/mL), together with smaller amounts of 4-hydroxybenzoic acid esters, benzoic acid, and salicylic acid.     

Quantitation of total folate in whole blood using LC-MS/MS

An accurate method for measuring whole blood total folate using liquid chromatography with tandem mass spectrometry is described and compared to GC/MS and a chemiluminescence assay. Whole blood from normal adults (n = 15) was fortified with a [(13)C(6)]para-aminobenzoic acid (pABA) internal standard and treated with 12.1 N hydrochloric acid at 110 degrees C for 4 h to hydrolyze all folates to pABA. Contaminants in the hydrolysate were adsorbed onto a C18 SPE cartridge. The eluate containing the folate catabolite pABA was partitioned into ethyl acetate and methylesterified with trimethylsilyldiazomethane. The methyl-pABA derivatives were quantified by positive-ion atmospheric pressure chemical ionization (APCI)LC-MS/MS. An isocratic mobile phase of acetonitrile-water (70:30) (v/v) on a C18 analytical column was used with a postcolumn reagent of 0.025% formic acid. The limit of quantitation for folate was 56.6 nmol/L RBC, and the limit of detection was 22.6 nmol/L RBC. Folate levels as determined by LC-MS/MS correlated well with the chemiluminescence assay and a GC/MS method. This new LC-MS/MS method provides enhanced sample throughput (n = 36 per day) as compared to GC/MS methods. LC-MS/MS will enable accurate measurements of red blood cell (RBC) folate in nutrition surveys and clinical trials.     

LC-MS/MS法研究甲磺隆和氯磺隆在不同条件土壤中的残留行为

在建立液相色谱三重串联四极杆质谱（LC-MS/MS）检测技术的基础上,采用PVC材料的圆柱形盆钵栽培方法,研究了不同土壤环境条件下甲磺隆和氯磺隆的残留特性。结果表明,甲磺隆和氯磺隆在水稻根际和非根际土壤中的残留量均呈不断下降趋势。处理后15d,根际和非根际土壤中两种农药残留量分别下降了44.7%和41.5%（甲磺隆）及38.7%和40.1%（氯磺隆）,根际和非根际的残留差异不显著（P〉0.05）;处理后31d,残留量分别下降了77.7%和64.8%（甲磺隆）及62.7%和50.1%（氯磺隆）,差异达极显著水平（P〈0.01）;处理后63d,残留量分别下降了96.4%和85.1%（甲磺隆）及90.0%和79.4%（氯磺隆）,残留差异达极显著水平（P〈0.01）。甲磺隆的降解趋势和氯磺隆基本一致,但下降幅度比氯磺隆大。二者在水稻根际和非根际土壤中的残留量均符合一级动力学方程式C=C0e-λ（tC代表浓度;C0代表初始浓度;t时间）,决定系数范围在0.9342～0.9957之间。在种植水稻的条件下,下层土的农药残留量呈先上升后下降的趋势,处理后数日内达最高点,之后不断下降,122d后低于检测限。水稻下层土残留的从无到有说明农药在土壤中的淋溶可能是水田环境农药残留降解的原因之一。干旱土壤条件下,土壤的淋溶作用不明显。说明水旱轮作有利于农药残留在土壤环境中的降解。     

Determination and confirmation of nitrofuran residues in honey using LC-MS/MS

A method was developed for the determination and confirmation of furazolidone, nitrofurazone, furaltadone, and nitrofurantoin as their side-chain residues in honey using liquid chromatography-tandem mass spectrometry (LC-MS/MS). An initial solid-phase extraction cleanup of the honey samples was followed by overnight hydrolysis and derivatization of the nitrofuran side-chain residues with 2-nitrobenzaldehyde. After pH adjustment and liquid-liquid extraction, the extracts were assayed by LC-MS/MS using electrospray ionization in the positive ion mode. The method was validated at concentrations ranging from 0.5 to 2.0 ppb with accuracies of 92-103% and coefficients of variation of < or =10%. The lowest calibration standard used (0.25 ppb) was defined as the limit of quantitation for all four nitrofuran side-chain residues. The extracts and standards were also used for confirmatory purposes. Honey from dosed beehives was assayed to study the stability of the nitrofuran residues and to demonstrate the effectiveness of the method.     

Development of stable isotope dilution assays for the simultaneous quantitation of biogenic amines and polyamines in foods by LC-MS/MS

Microbial amino acid metabolism may lead to substantial amounts of biogenic amines in either spontaneously fermented or spoiled foods. For products manufactured with starter cultures, it has been suggested that certain strains may produce higher amounts of such amines than others; however, to support efforts of food manufacturers in mitigating amine formation, reliable methods for amine quantitation are needed. Using 10 isotopically labeled biogenic amines as the internal standards, stable isotope dilution assays were developed for the quantitation of 12 biogenic amines and of the 2 polyamines, spermine and spermidine, in one LC-MS/MS run. Application of the method to several foods revealed high concentrations of, for example, tyramine and putrescine in salami and fermented cabbage, whereas histamine was highest in Parmesan cheese and fermented cabbage. On the other hand, ethanolamine was highest in red wine and Parmesan cheese. The results suggest that different amino acid decarboxylases are active in the respective foods depending on the microorganisms present. The polyamine spermine was highest in salami and tuna.