Phase Liquid Chromatography-tandem Mass Research Articles

Determination of eight phosphatidylethanol homologues in blood by reversed phase liquid chromatography–tandem mass spectrometry – How to avoid co-elution of phosphatidylethanols and unwanted phospholipids

Phosphatidylethanols (PEths) are specific, direct alcohol biomarkers with a substantially longer half-life than ethanol, and can be used to distinguish between heavy- and social drinking. More than forty PEth homologues have been detected in blood from heavy drinkers, and PEth 16:0/18:1 is the predominant one. Since PEths are phospholipids it can be difficult to isolate them from unwanted phospholipids during sample preparation. To minimize possible matrix effects it is therefore important to separate PEths from other phospholipids during LC-MS/MS analysis. In this study, we have investigated how the retention and chromatographic separation of eight PEth homologues and the phospholipid background are influenced by changes in mobile phase composition using two different LC columns, the Acquity BEH C18 column (50 × 2.1 mm ID, 1.7 µm particles) and the Kinetex biphenyl column (100 × 2.1 mm ID, 1.7 µm particles). Our findings show that the buffer concentration of the aqueous part of the mobile phase had a huge effect on the retention of PEth homologues and separation of PEths from unwanted phospholipids. By using a buffer-free mobile phase consisting of 0.025% ammonia in Type 1 water, pH 10.7, as solvent A and methanol as solvent B, all eight PEth homologues were separated from both the early eluting lyso-phospholipids and the later eluting phospholipids with two fatty chains using the BEH C18 column. The knowledge obtained in this study can be of great importance for those seeking to develop reliable and robust bioanalytical LC-MS/MS methods for determination of PEth homologues.

A sensitive LC–MS/MS method for isomer separation and quantitative determination of 51 pyrrolizidine alkaloids and two tropane alkaloids in cow’s milk

1,2-Unsaturated pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO), and tropane alkaloids (TA) are toxic secondary plant metabolites. Their possible transfer into the milk of dairy cows has been studied in feeding trials; however, only few data on the occurrence of these toxins in milk are available. In this study, the development of a sensitive analytical approach for the simultaneous detection and quantification of a broad range of 54 PA/PANO as well as of the TA atropine and scopolamine in milk of dairy cows is presented. The method optimisation focused on sensitivity and separation of PA/PANO isomers. Milk samples were extracted using liquid–liquid extraction with aqueous formic acid and n-hexane, followed by a cation-exchange solid-phase extraction for purification. Reversed phase liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis was performed using alkaline solvent conditions. Validation proved low limits of detection and quantification of 0.005 to 0.054 µg/L and of 0.009 to 0.123 µg/L, respectively. For 51 of the 54 tested PA/PANO and both TA, the recovery rates ranged from 64 to 127% with repeatability (RSDr) values below 15% at concentration levels of 0.05 and 0.50 µg/L and below 8% at a concentration level of 3.00 µg/L. Only three PANO did not match the validation criteria and were therefore regarded as semiquantitative. The final method was applied to 15 milk samples obtained from milk vending stations at farms and from local marketers in Bavaria, Germany. In three of the milk samples, traces of PA were detected.Graphical abstract

Metabolomic navigated Citrus waste repurposing to restore amino acids disorder in neural lesion

The fruit juice food industry produces huge waste annually, mainly Citrus peel and seeds. We investigated their chemical composition using hydrophilic interaction chromatography (HILIC-) and reverse phase liquid chromatography tandem mass spectrometry (RPLC-MS/MS), revealing 277 compounds, mainly containing flavonoids and limonoids. As the primary representative component in Citrus waste, limonin was selected to be explored new bio-functions. We applied Zebrafish larvae to study the metabolomic response invoked by limonin. The differential metabolites (DMs) varied depending on the exposing concentration of limonin. Enrichment analysis indicated that the identified DMs related to inflammation and neurologic disorders, including epilepsy which were newly discovered for limonoids and Citrus waste. Limonin was found to restore amino acids disorder to take neuroprotection against epilepsy. Our findings provided a new bio-function and purpose for Citrus waste and limonoids. Also, we demonstrated a concise case to repurpose food waste for new applications under metabolome investigation.

Method development and validation for analysis of phenylalanine, 4-hydroxyphenyllactic acid and 4-hydroxyphenylpyruvic acid in serum and urine.

Alkaptonuria (AKU) is a rare debilitating autosomal recessive disorder of tyrosine (TYR) metabolism which results in a deficiency of the enzyme homogentisate 1,2‐dioxygenase activity. Several studies have reported the metabolic changes in homogentisic acid (HGA) concentrations and subsequent deposition of an ochronotic pigment in connective tissues, especially cartilage. Treatment with nitisinone (NTBC) reduces urinary and circulating HGA, but its mode of action results in hypertyrosinaemia. The effect of NTBC on other metabolites in the TYR pathway has not been reported. Modification of the current reverse phase liquid chromatography tandem mass spectrometry methods for serum and urine to include phenylalanine (PHE), hydroxyphenyllactate (HPLA) and hydroxyphenylpyruvate (HPPA) has been validated. HPPA and HPLA (negative ionisation) eluted at 2.8 and 2.9 min respectively on an Atlantis C18 column with PHE (positive ionisation) eluting earlier at 2.4 min. Intra‐ and inter‐assay accuracy was between 96.3% and 100.3% for PHE; 96.6% and 110.5% for HPLA and 95.0% and 107.8% for HPPA in both urine and serum. Precision, both inter‐ and intra‐assay, was <10% for all analytes in both serum and urine. No significant issues with carry‐over, stability or matrix interferences were seen in either the urine or serum assays. Measurement of serum and urine from AKU patients has demonstrated a robust, fully validated assay, appropriate for monitoring of patients with AKU and for demonstrating metabolite changes, following NTBC therapy.

Electron Transfer Dissociation Parameter Optimization Using Design of Experiments Increases Sequence Coverage of Monoclonal Antibodies.

Middle-down analysis of monoclonal antibodies (mAbs) by tandem mass spectrometry (MS2) can provide detailed insight into their primary structure with minimal sample preparation. The middle-down approach uses an enzyme to cleave mAbs into Fc/2, LC, and Fd subunits that are then analyzed by reversed phase liquid chromatography tandem mass spectrometry (RPLC-MS2). As maximum sequence coverage is desired to obtain meaningful structural information at the subunit level, a host of dissociation methods have been developed, and sometimes combined, to bolster fragmentation and increase the number of identified fragments. Here, we present a design of experiments (DOE) approach to optimize MS2 parameters, in particular those that may influence electron transfer dissociation (ETD) efficiency to increase the sequence coverage of antibody subunits. Applying this approach to the NIST monoclonal antibody standard (NISTmAb) using three RPLC-MS2 runs resulted in high sequence coverages of 67%, 67%, and 52% for Fc/2, LC, and Fd subunits, respectively. In addition, we apply this DOE strategy to model the parameters required to maximize the number of fragments produced in "low", "medium", and "high" mass ranges, which ultimately resulted in even higher sequence coverages of NISTmAb subunits (75%, 78%, and 64% for Fc/2, LC, and Fd subunits, respectively). The DOE approach provides high sequence coverage percentages utilizing only one fragmentation method, ETD, and could be extended to other state-of-the-art techniques that combine multiple fragmentation mechanisms to increase coverage.

Temporal proteomic profiling reveals changes that support Burkholderia biofilms.

Melioidosis associated with opportunistic pathogen Burkholderia pseudomallei imparts a huge medical burden in Southeast Asia and Australia. At present there is no available human vaccine that protects against B. pseudomallei infection and antibiotic treatments are limited particularly for drug-resistant strains and bacteria in biofilm forms. Biofilm forming bacteria exhibit phenotypic features drastically different to their planktonic states, often exhibiting a diminished response to antimicrobial therapies. Our earlier work on global profiling of bacterial biofilms using transcriptomics and proteomics revealed transcript-decoupled protein abundance in bacterial biofilms. Here we employed reverse phase liquid chromatography tandem mass spectrometry (LC-MS/MS) to deduce temporal proteomic differences in planktonic and biofilm forms of Burkholderia thailandensis, which is weakly surrogate model of pathogenic B. pseudomallei as sharing a key element in genomic similarity. The proteomic analysis of B. thailandensis in biofilm versus planktonic states revealed that proteome changes support biofilm survival through decreased abundance of metabolic proteins while increased abundance of stress-related proteins. Interestingly, the protein abundance including for the transcription protein TEX, outer periplasmic TolB protein, and the exopolyphosphatase reveal adaption in bacterial biofilms that facilitate antibiotic tolerance through a non-specific mechanism. The present proteomics study of B. thailandensis biofilms provides a global snapshot of protein abundance differences and antimicrobial sensitivities in planktonic and sessile bacteria.

Construction of on-line supercritical fluid extraction with reverse phase liquid chromatography–tandem mass spectrometry for the determination of capsaicin

A novel on-line system composed of supercritical fluid extraction (SFE), dilution line and reverse phase liquid chromatography/mass spectrometry (RPLC/MS) was constructed for on-line extraction and reverse phase separation of some fat-soluble components in foods. Three columns including a trap column, concentration column and analytical column were used for trapping the fat-soluble components, on-line enrichment and reverse phase separation, respectively. Capsaicinoids were on-line extracted by a CO2 supercritical fluid, then concentrated and separated by using the C18 columns, finally detected by mass spectrometry (MS). Capsaicin eluted at 10.1 min and limit of detection (LOD, S/N = 3) for the standard solution is 0.55 pg. The linearity was calculated with a value of coefficient of determination (R2) ≥0.998 in the range of 1.1–8.5ng. Concentrations of capsaicin in the green, yellow, and red bell peppers were determined to be 60.33ng/g, 31.79 ng/g, 35.38ng/g, respectively.

Enantioselective degradation and transformation of the chiral fungicide prothioconazole and its chiral metabolite in soils

Prothioconazole is a widely used chiral triazole fungicide. In this work, the enantioselective degradation and transformation of prothioconazole and its chiral metabolite prothioconazole-desthio in five kinds of soils were investigated under native and sterile conditions using reversed phase liquid chromatography tandem mass spectrometry with a Lux-cellulose-1 column. The results showed that an enantioselective degradation was observed with R-prothioconazole preferentially degraded in the five soils and enantiomeric fraction values that ranged from 0.32 to 0.41 under native conditions. Furthermore, the major metabolite prothioconazole-desthio was formed rapidly during prothioconazole dissipation. The prothioconazole-desthio enantiomers were degraded slowly, and there was a slight enantioselectivity with enantiomeric fraction values that ranged from 0.45 to 0.51 in the Nanjing and Jilin soils. Under sterile conditions, prothioconazole and its metabolite enantiomers were more slowly degraded with no enantioselectivity. The result of the incubation experiment with single enantiomers verified that R- and S-prothioconazole were transformed to R- and S-prothioconazole-desthio, respectively. No enantiomerization for prothioconazole and its chiral metabolite was observed. In addition, the excellent correlation between organic matter content and degradation rate indicated that organic matter could promote the degradation of prothioconazole and its metabolite enantiomers. The data in this study provide the experimental evidence of the stereoselective degradation and metabolism of both prothioconazole and its chiral metabolite in the environment.

Development and validation of a bioanalytical method for quantification of LNA-i-miR-221, a 13-mer oligonucleotide, in rat plasma using LC–MS/MS

LNA-i-miR-221, a 13-mer oligonucleotide, is a new miR-221 inhibitor that could be used as a novel drug for multiple myeloma. Herein, an ion-pair reversed phase liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the quantification of LNA-i-miR-221 in rat plasma. Plasma samples were prepared with an initial phenol/chloroform/isoamyl alcohol liquid–liquid extraction followed by a solid phase extraction. Chromatographic separation was performed with a gradient system on a HALO C18 column using hexafluoro-2-propanol/triethylamine buffer and methanol as mobile phase at a flow rate of 0.4 mL/min. Under these conditions LNA-i-miR-221 and the analogue internal standard are co-eluted at 1.2 min. The detection was carried out in multiple reaction monitoring (MRM) mode using a negative electrospray ionization (ESI) interface. The assay showed a good linearity within the calibration range 10–10000 ng/mL. The precision, accuracy, and recovery values were found to be <15% (<20% at LLOQ), 100 ± 15%, and 97.6–103.7%, respectively. This method was successfully applied to measure the concentrations of LNA-i-miR-221 in plasma samples following the intravenous administration during a 4-week toxicity study in rats.

Chiral Analysis of Methamphetamine in Oral Fluid Samples: A Method to Distinguish Licit from Illicit Drug Use.

Methamphetamine (MAMP) is a popular illicit drug abused for its central nervous system stimulating effects. MAMP is also used therapeutically in the treatment of overeating disorders, narcolepsy, attention deficit disorder, in over-the-counter (OTC) products to ease nasal congestion. MAMP exists in two enantiomeric forms, dextrorotary (d-MAMP) or levorotary (l-MAMP). The compounds are similar in chemical structure, simply differing in the orientation of functional groups around the asymmetric carbon. In part because of the availability of l-MAMP in OTC nasal inhalers, forensic guidelines require a sample to contain greater than 20% d-MAMP to consider illicit drug use when interpreting results. Standard analytical methods readily detect MAMP in biological specimens but cannot resolve the enantiomeric composition of the sample. Specialized analytical techniques based on chiral separation of the enantiomers are required to differentiate d-MAMP from l-MAMP. Our laboratory sought to develop and validate a method for the analysis of oral fluid specimens for d/l-MAMP using a chiral derivatizing agent and traditional reverse phase liquid chromatography tandem mass spectrometry (LC-MS-MS). MAMP was extracted from dilute oral fluid samples using Strata-XC solid phase extraction (SPE) cartridges and derivatized with Marfey's reagent. Chromatographic separation was achieved using Zorbax Eclipse Plus C18 columns. Linearity, accuracy and precision, recovery, matrix effects and specificity of the method were all within acceptable criteria. Intraday accuracy ranged from 93.3 to 103.4% and precision 0.1 to 1.6%. Interday accuracy ranged from 90.0 to 103.4% and precision 3.8 to 11.6%. Finally, having previously tested positive for MAMP using non-chiral analysis, 256 de-identified authentic oral fluid samples were analyzed using this validated method. 98% of all samples tested positive for d-MAMP at greater than 20%.

Determination of 27 new fungicides in cereals, vegetables and fruits by dispersive solid phase extraction and liquid chromatography-tandem mass spectrometry

Determination of 27 new fungicides in cereals, vegetables and fruits by dispersive solid phase extraction and liquid chromatography-tandem mass spectrometry

Novel Two-Dimensional Liquid Chromatography–Tandem Mass Spectrometry for the Analysis of Twenty Antibiotics Residues in Dairy Products

A novel two-dimensional hydrophilic interaction chromatography/reversed phase liquid chromatography–tandem mass spectrometry system was developed to analyze seven categories of antibiotics residues in dairy products. The seven categories were β-lactams, tetracyclines, macrolides, aminoglycosides, amphenicols, quinolones, and sulphonamides, and involved 20 antibiotics. Samples of milk powder or milk samples were extracted by mixed matrix solid phase dispersion using C18 and CN material. The analytes were eluted by acetonitrile and water, and were dried with rotary evaporation. Finally, the residues were dissolved with mobile phase and then analyzed. Under sample pretreatment conditions, chromatographic and mass spectrometric parameters were optimized. Twenty analytes showed good linearities with correlation coefficients of 0.9945 ~ 0.9998. The limits of detection and quantification for milk powder and milk samples were 0.10 ~ 2.40 and 0.33 ~ 7.92 μg/kg, respectively. The proposed method has been applied to the determination of antibiotics residues in milk powder and milk samples.

Gender-related pharmacokinetics and bioavailability of a novel anticancer chalcone, cardamonin, in rats determined by liquid chromatography tandem mass spectrometry.

A reversed phase liquid chromatography tandem mass spectrometry method was developed and validated for quantification of cardamonin, a potential anticancer chalcone, in rat serum. Curcumin was used as an internal standard. Following liquid-liquid extraction using n-hexane and ethyl acetate (60:40, v/v), the processed samples were chromatographed on a C18 column using acetonitrile and ammonium acetate buffer (0.01 M, pH 4.5) (85:15, v/v) as mobile phase at a flow rate of 0.6 mL min(-1). Mass spectrometric detection was performed in the negative electrospray ionization mode by multiple reaction monitoring (m/z 269→122 and 367→217 for cardamonin and curcumin, respectively). The method was validated in terms of selectivity, accuracy, precision, sensitivity, reproducibility, dilution integrity and stability. The linearity was established in the range of 1-200 ng mL(-1) (r≥0.999). The recovery of cardamonin from spiked serum was always >90%. The intra- and inter-day precision (%RSD) and accuracy (%bias) were well within the acceptable limits. The method was applied for single oral and intravenous dose pharmacokinetics in male and female Sprague Dawley rats. Following oral dose, cardamonin showed peak serum concentration that occurred at ∼2 h with very low bioavailability in both male (0.6%) and female (4.8%) rats. Cardamonin exhibited a significant gender influence on pharmacokinetics and bioavailability in rats.

Serum markers in alkaptonuria: simultaneous analysis of homogentisic acid, tyrosine and nitisinone by liquid chromatography tandem mass spectrometry.

Alkaptonuria is a rare debilitating autosomal recessive disorder of tyrosine metabolism, where deficiency of homogentisate 1,2-dioxygenase results in increased homogentisic acid. Homogentisic acid is deposited as an ochronotic pigment in connective tissues, especially cartilage, leading to a severe early onset form of osteoarthritis, increased renal and prostatic stone formation and hardening of heart vessels. Treatment with the orphan drug, nitisinone, an inhibitor of 4-hydroxyphenylpyruvate dioxygenase has been shown to reduce urinary excretion of homogentisic acid. A reverse phase liquid chromatography tandem mass spectrometry method has been developed to simultaneously analyse serum homogentisic acid, tyrosine and nitisinone. Using matrix-matched calibration standards, two product ion transitions were identified for each compound (homogentisic acid, tyrosine, nitisinone) and their respective isotopically labelled internal standards ((13)C6-homogentisic acid, d2-tyrosine, (13)C6-nitisinone). Intrabatch accuracy was 94-108% for homogentisic acid, 95-109% for tyrosine and 89-106% for nitisinone; interbatch accuracy (n = 20) was 88-108% for homogentisic acid, 91-104% for tyrosine and 88-103% for nitisinone. Precision, both intra- and interbatch were <12% for homogentisic acid and tyrosine, and <10% for nitisinone. Matrix effects observed with acidified serum were normalized by the internal standard (<10% coefficient of variation). Homogentisic acid, tyrosine and nitisinone proved stable after 24 h at room temp, three freeze-thaw cycles and 24 h at 4℃. The assay was linear to 500μmol/L homogentisic acid, 2000μmol/L tyrosine and 10μmol/L nitisinone; increased range was not required for clinical samples and no carryover was observed. The method developed and validated shows good precision, accuracy and linearity appropriate for the monitoring of alkaptonuria patients, pre- and post-nitisinone therapy.

Determination of 19 Acidic Pesticide Residues in Fruit by Dispersive Solid Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry

建立了同时检测葡萄、苹果和桔子等水果中2,4-滴等19种酸性农药的分散固相萃取-液相色谱-串联质谱(HPLC-MS/MS)方法。葡萄、苹果和桔子样品经乙酸-乙腈(1∶99,V/V)提取后,C18分散固相萃取净化,采用反相C18色谱柱分离。以0.1%甲酸和0.1%甲酸-乙腈溶液作为流动相进行梯度洗脱,采用多反应监测离子模式进行定性分析,基质标准曲线外标法进行定量分析,线性范围在0.01~0.2 mg/kg之间。在0.02,0.05和0.1 mg/kg添加水平下,19种酸性农药的回收率为70.3%～105.3%;相对标准偏差为0.37%～10.9%。本方法的定量限为0.005～0.02 mg/kg...

Bidirectional Chiral Inversion of Trantinterol Enantiomers After Separate Doses to Rats

The chiral inversion and pharmacokinetics of two enantiomers of trantinterol, a new β2 agonist, were studied in rats dosed (+)- or (-)-trantinterol separately. Plasma concentrations of (+)- and (-)-trantinterol were measured by chiral stationary phase liquid chromatography tandem mass spectroscopy (LC-MS/MS). The apparent inversion ratio was calculated as the ratio of AUC0-t of (-)-trantinterol or (+)-trantinterol inverted from their antipodes to the sum of the AUC0-t of (-)- and (+)-trantinterol. Following single intravenous administration, both given enantiomers declined in similar plasma concentrations, suggesting that the two enantiomers have approximately the same disposition kinetics by the route of intravenous administration. However, after single oral administration, plasma concentrations of uninverted (-)-trantinterol at many timepoints were significantly higher than those of uninverted (+)-trantinterol, suggesting that the two enantiomers undergo apparently different absorption or metabolism after oral administration. Significant bidirectional chiral inversion occurred after intravenous and oral administration of (+)- or (-)-trantinterol. After dosing with optically pure enantiomer, the concentration of the administered enantiomer predominated in vivo. The AUC0-36 of (+)-trantinterol after intravenous and oral dosing of (-)-trantinterol were 16.6 ± 5.2 and 33.3 ± 16%, respectively of those of total [(+) + (-)] trantinterol. The AUC0-36 of (-)-trantinterol after intravenous and oral dosing of (+)-trantinterol were 19.6 ± 8.8 and 37.9 ± 4.5%, respectively, of those of total [(-) + (+)] trantinterol. After intravenous administration of (+)- and (-)-trantinterol the chiral inversion ratios of the two enantiomers were not significantly different and similar results were found for oral administration. The extent of chiral inversion after intravenous administration was apparently lower, indicating that the bidirectional chiral inversion was not only systemic but also presystemic.

On-line two-dimensional capillary strong anion exchange/reversed phase liquid chromatography–tandem mass spectrometry for comprehensive lipid analysis

An on-line two-dimensional liquid chromatography method was developed for comprehensive lipid profiling by coupling strong anion exchange (SAX) and nanoflow reversed-phase liquid chromatography (nRPLC) prior to electrospray ionization-tandem mass spectrometry (2D-SAX/nRPLC–ESI-MS/MS). Lipids can be classified into four different types according to the electrical propensities of the lipids: anionic, weak anionic, neutral polar, and special lipids. In 2D-SAX/nRPLC, various lipids can be fractionated in the first dimension (SAX: 5μm to 100Å, 5.0cm×75μm i.d.) by step elution (methanol and salt solution), followed by the molecular separation of lipids in the second dimension (RP: 3μm to 100Å, 7.0cm×75μm i.d.) with binary gradient LC. Since the elution of lipids from SAX can be achieved with a very small volume of eluent delivered from an autosampler, it can be simply implemented with an LC–ESI-MS instrument for full automation, and the salt step elution, including the two-step injection procedure, can be used for the selective analysis of the desired lipid fraction. For nRPLC–ESI-MS/MS run in either positive or negative ion mode, a common ionization modifier (0.05% ammonium hydroxide with 5mM ammonium formate) was introduced into the binary mobile phase solutions so that 2D-LC–MS could be operated in both ion modes without changing the mobile phase solutions. The developed on-line 2D-SAX/nRPLC–ESI-MS/MS was evaluated with 22 different standard lipids for the optimization of the salt step elution and was applied to a healthy human plasma lipid extract, resulting in the identification of a total of 303 plasma lipids, including 14 different classes.

Simultaneous Extraction and Determination of Three Microcystins from Soil Using Solid Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry

An analytical method was developed for the simultaneous extraction and determination of three microcystins(MCs) from soils using solid phase extraction and liquid chromatography tandem mass spectrometry.Soil sample was firstly vortex extracted by EDTA-sodium pyrophosphate,then purified and concentrated by Sep-Pak C18 SPE cartridge.Qualitative and quantitative analysis was carried out under the MRM mode after the chromatography separation.The range of recoveries for MC-LR,MC-RR and MC-YR in the soil matrix was 72.6%-97.4%,54.9%-62.8% and 69.0%-90.7% with 4.3%-16.9% of relative standard deviation respectively in three different spiked concentrations.The limits of quantification were 0.25,0.25 and 0.5 μg/kg for MC-LR,MC-RR and MC-YR,respectively.The method was used to analyse arable soil samples collected around Dianchi Lake,Yunnan Province.Results showed the microcystins in the arable soil were detected with sum concentration ranged from 1.43 to 21.29μg/kg.

Simultaneous determination of carbamate insecticides and mycotoxins in cereals by reversed phase liquid chromatography tandem mass spectrometry using a quick, easy, cheap, effective, rugged and safe extraction procedure

A simple, sensitive and reliable analytical method was developed for the simultaneous determination of 22 carbamate insecticides and 17 mycotoxins in cereals by ultra high performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS). Carbamates and mycotoxins were extracted from cereal samples using a QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) procedure without any further clean-up step. The extract was diluted with water containing 0.1% formic acid and 5.0mM ammonium acetate, and analyzed by LC-MS/MS on a Waters Acquity BEH C(18) column with water (0.1% formic acid, 0.50mM ammonium acetate)/methanol as mobile phase with gradient elution. Matrix-matched calibration was used for quantification. Blank samples (rice, wheat and corn) were fortified at 5, 10 and 50 μg/kg except for five zearalenonic compounds at 25, 50 and 250 μg/kg, and recoveries were in the range of 70-120%. Relative standard deviations were lower than 20% in all cases. The LOQ values were in the range of 0.20-29.7 μg/kg. The method is suitable for the simultaneous determination of carbamate insecticides and mycotoxins in cereals. The total time required for the analysis of one sample, including sample preparation, was about 35 min.

Determination of phenylethanolamine A in animal hair, tissues and feeds by reversed phase liquid chromatography tandem mass spectrometry with QuEChERS

A simple, sensitive and reliable analytical method was developed for the determination of a new beta-agonist phenylethanolamine A in animal hair, tissues and animal feeds by ultra high performance liquid chromatography–positive electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS) with QuEChERS. Samples were extracted with acetonitrile/water (80:20, v/v). The extract was purified through QuEChERS method, then was dried with nitrogen and residues were redissolved in mobile phase for hair sample or directly diluted with 0.1% formic acid in water for other samples, and analyzed by LC–MS/MS on a Waters Acquity BEH C18 column with 0.1% formic acid in water/methanol as mobile phase with gradient elution. The samples were quantified using phenylethanolamine A-D3 as internal standards. The proposed method was validated according to the European Commission Decision 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), recovery, precision, linearity, robustness and stability. The CCα values ranged from 0.10 to 0.26μg/kg. The CCβ values ranged from 0.20 to 0.37μg/kg. The mean recoveries of 95.4–108.9% with intra-day CVs of 2.2–5.6% and inter-day CVs of 3.1–6.2% were obtained. The method is demonstrated to be suitable for the determination of phenylethanolamine A in animal hair, tissues and animal feeds. The total time required for the analysis of one sample except animal hair sample, including sample preparation, was about 25min.