Detection of 13 mycotoxins in feed using modified QuEChERS with dispersive magnetic materials and UHPLC–MS/MS

An analytical method for the simultaneous determination of 13 mycotoxins in feed by magnetic dispersive solid‐phase extraction combined with ultra‐high performance liquid chromatography and tandem mass spectrometry was developed. The samples were extracted with acetonitrile/water (80:20, v/v, containing 3% acetic acid), and separated by centrifugation after salting‐out, and then treated with magnetic adsorbents to remove interferences. The separation of target mycotoxins was performed on an ACQUITY UPLC HSS T3 column using a mobile phase consisting of 1 mmol/L ammonium acetate with 0.1% formic acid and methanol by gradient elution. Good linearities for the 13 mycotoxins were achieved with correlation coefficients over 0.99, and the recoveries of mycotoxins were in the range of 89.3–112.6% at spiking at levels of 5, 20, and 100 μg/kg, with relative standard deviations of 0.9–10.4%. Based on the functional magnetic materials (MDN@Fe₃O₄, PSA@Fe₃O₄, ZrO_2@Fe₃O₄) applied in dispersive solid‐phase extraction, the pretreatment process is more convenient and it is beneficial to reduce the experimental cost by reusing the recycled magnetic materials. It is a simple, rapid, and environmentally friendly analytical method for the determination of mycotoxins in feed.     

Multiplug filtration clean‐up with multiwalled carbon nanotubes in the analysis of pesticide residues using LC–ESI‐MS/MS

A novel design for a rapid clean‐up method was developed for the analysis of pesticide residues in fruit and vegetables followed by LC–ESI‐MS/MS. The acetonitrile‐based sample extraction technique was used to obtain the extracts, and further clean‐up was carried out by applying the streamlined procedure on a multiplug filtration clean‐up column coupled with a syringe. The sorbent used for clean‐up in this research is multiwalled carbon nanotubes, which was mixed with anhydrous magnesium sulfate to remove water from the extracts. This method was validated on 40 representative pesticides and apple, cabbage, and potato sample matrices spiked at two concentration levels of 10 and 100 μg/kg. It exhibited recoveries between 71 and 117% for most pesticides with RSDs < 15%. Matrix‐matched calibrations were performed with the coefficients of determination >0.995 for most studied pesticides between concentration levels of 10–500 μg/L. The LOQs for 40 pesticides ranged from 2 to 50 μg/kg. The developed method was successfully applied to the determination of pesticide residues in market fruit and vegetable samples.     

Detection and characterization of prednisolone metabolites in human urine by LC‐MS/MS

Glucocorticosteroids are prohibited in sports when used by systemic administrations (e.g. oral), whereas they are allowed using other administration ways. Strategies to discriminate between administrations routes have to be developed by doping control laboratories. For this reason, the metabolism of prednisolone (PRED) was studied using liquid chromatography coupled to tandem mass spectrometry. A single oral (10 mg) dose of PRED was administered to two healthy male volunteers. Urine samples were collected up to 6 days after administration. Samples were hydrolyzed with β‐glucuronidase and subjected to liquid–liquid extraction with ethyl acetate in alkaline conditions. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry. Precursor ion scan methods (m/z 77, 91, 105, 121, 147 and 171) in positive ionization and neutral loss scan methods (76 and 94 Da) in negative ionization modes were applied for the open detection of PRED metabolites. Using these methods, PRED parent compound plus 20 metabolites were detected. PRED and 11 metabolites were characterized by comparison with standards of the compounds (PRED, prednisone, 20β‐dihydro‐PRED and 20α‐dihydro‐PRED, 20β‐dihydro‐prednisone and 20α‐dihydro‐prednisone, 6β‐hydroxy‐PRED and 6α‐hydroxy‐PRED, 20β isomers and 20α isomers of 6β,11β,17α,20,21‐pentahydroxypregnan‐1,4‐diene‐3‐one, 6α,11β,17α,20β,21‐pentahydroxypregnan‐1,4‐diene‐3‐one and Δ⁶‐PRED). Using mass spectrometric data, feasible structures were proposed for seven of the remaining nine detected metabolites, including several 6‐hydroxy‐metabolites. Eleven of the characterized metabolites have not been previously described. Maximum excretion rates for PRED metabolites were achieved in first 24 h; however, most of the metabolites were still detectable in the last collected samples (day 6). Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of avoparcin in animal tissues and milk using LC‐ESI‐MS/MS and tandem‐SPE

A highly sensitive and selective method using LC‐ESI‐MS/MS and tandem‐SPE was developed to detect trace amounts of avoparcin (AV) antibiotics in animal tissues and milk. Data acquisition using MS/MS was achieved by applying multiple reaction monitoring of the product ions of [M + 3H]³⁺ and the major product ions of AV‐α and ‐β at m/z 637 → 86/113/130 and m/z 649 → 86/113/130 in ESI(+) mode. The calculated instrumental LODs were 3 ng/mL. The sample preparation was described that the extraction using 5% TFA and the tandem‐SPE with an ion‐exchange (SAX) and InertSep C18‐A cartridge clean‐up enable us to determine AV in samples. Ion suppression was decreased by concentration rates of each sample solution. These SPE concentration levels could be used to detect quantities of 5 ppb (milk), 10 ppb (beef), and 25 ppb (chicken muscle and liver). The matrix matching calibration graphs obtained for both AV‐α (r >0.996) and ‐β (r >0.998) from animal tissues and milk were linear over the calibration ranges. AV recovery from samples was higher than 73.3% and the RSD was less than 12.0% (n = 5).     

免疫亲和柱净化-液相色谱-串联质谱法测定中药材中的14种真菌毒素

依次采用磷酸盐缓冲液(PBS)和甲醇-PBS溶液提取样品,以多功能免疫亲和柱净化,采用液相色谱-串联四极杆质谱检测,可同时测定中药材中的黄曲霉毒素B1、黄曲霉毒素B2、黄曲霉毒素G1、黄曲霉毒素G2、伏马毒素B1、伏马毒素B2、T-2毒素、HT-2毒素、赭曲霉毒素A(OTA)、玉米赤霉烯酮等14种真菌毒素。优化条件下,真菌毒素的定量限(LOQ)为1～5 μg/kg, 4种中药材基质(人参、桔梗、板蓝根、麦门冬)中3个不同添加水平的平均回收率(n=6)为71.9%～99.7%,相对标准偏差(RSD)为4.8%～15.8%。该方法的检测速度快,中药材复杂基质的干扰较少,结果准确、可靠,定量限可满足国内外中药材真菌毒素相关限量的要求。     

Fast ultrasound‐assisted extraction combined with LC–MS/MS of perfluorinated compounds in manure

A fast analytical method for the determination of perfluorinated compounds in poultry manure by LC–MS/MS was developed. The extraction was carried out by ultrasound‐assisted extraction of 1 g of sample, during 2 × 15 min using low volume (5.5 mL) of a mixture of methanol and acetonitrile. An efficient extraction of perfluoroalkyl carboxylates, perfluoroalkyl sulfonates, and perfluoroalkyl sulfonamides from poultry manure was obtained with recoveries higher than 81%. The cleanup of extracts was carried out by dispersive SPE. The validation of the proposed method showed the suitability of this procedure to determine perfluorinated compounds in poultry manure with detection limits in the range of 0.44–2.12 ng/g, depending on the target compound. In comparison with previously published methods, the miniaturization of the sample preparation method with ultrasound‐assisted extraction together with the use of a core‐shell column permit a lower consumption of organic solvents and a fast analysis of perfluorinated compounds. Manure samples obtained from Spanish commercial farms were analyzed and low perfluorinated compounds levels were found, which may be originated by dietary or environmental exposure. The highest concentrations measured corresponded to the perfluoroalkyl sulfonates, which varied from 8.2 to 35.9 ng/g.     

Simultaneous quantification of atractyloside and carboxyatractyloside in rat plasma by LC‐MS/MS: Application to a pharmacokinetic study after oral administration of Xanthii Fructus extract

Xanthii Fructus is extensively used as an herbal medicine. Ingestion of this herb is associated with severe hepatotoxicity and nephrotoxicity. Atractyloside and carboxyatractyloside are two dominative toxic constituents in Xanthii Fructus. However, their pharmacokinetic study is lacking. In this study, a novel high‐performance liquid chromatography‐tandem mass spectrometry method was developed to simultaneously quantify the rat plasma concentrations of atractyloside and carboxyatractyloside. After protein precipitation, the analytes were chromatographic separated on a ZORBAX Eclipse Plus column (2.1 × 150 mm id, 5 µm) under gradient elute. In the negative electrospray ionization mode, the transitions at m/z 725.3→645.4 for atractyloside, m/z 769.3→689.4 for carboxyatractyloside, and m/z 479.2→121.1 for paeoniflorin (the internal standard) were acquired by multiple reaction monitoring. This analytical method showed good linearity over 1–500 ng/mL for atractyloside and 2–500 ng/mL for carboxyatractyloside with acceptable precision and accuracy. No matrix effect, instability and carryover occurred in the analysis procedure. The extraction recoveries were greater than 85.0%. This method was applied to a preliminary pharmacokinetic study by orally administering Xanthii Fructus extract (9 g/kg) to rats, which was useful to evaluate the role of these two compounds in Xanthii Fructus‐induced toxicity.     

Benzylisoquinoline alkaloid analysis using high‐resolution Orbitrap LC‐MSn

Benzylisoquinoline alkaloids (BIAs) have profound implications on human health owing to their potent pharmacological properties. Notable naturally occurring BIAs are the narcotic analgesics morphine, the cough suppressant codeine, the potential anticancer drug noscapine, the muscle relaxant papaverine, and the antimicrobial sanguinarine, all of which are produced in opium poppy (Papaver somniferum). Thebaine, an intermediate in the biosynthesis of codeine and morphine, is used in the manufacture of semisynthetic opiates, including oxycodone and naloxone. As the only commercial source of pharmaceutical opiates, opium poppy has been the focus of considerable research to understand BIA metabolism in the plant. The elucidation of several BIA biosynthetic pathways has enabled the development of synthetic biology platforms aimed at the alternative commercial production of valuable phytochemicals in microorganisms. The detection and identification of BIA pathway products and intermediates in complex extracts is essential for the continuing advancement of research in plant specialized metabolism and microbial synthetic biology. Herein, we report the use of liquid chromatography coupled with linear trap quadrupole and high‐resolution Orbitrap multistage mass spectrometry to characterize 44 authentic BIAs using collision‐induced dissociation (CID), higher‐energy collisional dissociation (HCD), and pulsed Q collision‐induced dissociation (PQD) MS² fragmentation, with MS² CID followed by MS³ and MS⁴ fragmentation. Our deep library of diagnostic spectral data constitutes a valuable resource for BIAs identification. In addition, we identified 22 BIAs in opium poppy latex and roots extracts.     

Development of rapid and high‐throughput LC–MS/MS method for quantification of olprinone in rabbit plasma

A simple, highly sensitive and rapid method for quantification of olprinone (phosphodiesterase 3 inhibitor) in rabbit plasma using liquid chromatography–tandem mass spectrometry with electrospray was developed. An aliquot of 50 μL of plasma sample was cleaned up and extracted using Ostro? 96‐well plate followed by dilution. Chromatographic separation of olprinone and olprinone‐d3 was carried out on a CORTECS^® T3 column within 3 min. Detection was achieved using a triple quadrupole mass spectrometer employing electrospray ionization operated in positive ion multiple reaction monitoring mode using the transitions m/z 251.07 → m/z 155.06 and m/z 254.21 → m/z 158.10 for olprinone and olprinone‐d3, respectively. The method was validated according to US Food and Drug Administration guideline for bioanalytical methods, and showed excellent linearity in the range 10.0–2000.0 ng/mL with coefficient of determination >0.99. The intra‐ and inter‐day precisions (CV) were <5.1% and the accuracies were within the range 99.7–103.2% at all quality control concentrations. Furthermore, olprinone was stable under various stability conditions. The developed method was used for quantification of olprinone in rabbit plasma after its intravenous administration at the dose of 1 mg/kg in order to better understand the metabolism of olprinone in a rabbit model of lung injury.     

Identification of short‐chain poly‐3‐hydroxybutyrates in Saiga horn extracts using LC–MS/MS

Saiga horn extracts were analyzed with the goal of obtaining new information about compounds present in it. The purpose of this study is to find synthetic alternatives to Saiga horn extract, which is used in traditional Chinese medicine, by identifying potentially biologically active compounds in the extracts. Using high‐performance liquid chromatography coupled with high‐resolution mass spectrometry, we have been able to identify a series of short‐chain polyhydroxybutyrates in alcoholic extracts of Saiga horn. Optimized high‐performance liquid chromatography coupled with tandem mass spectrometry methods for analysis of short‐chain poly‐3‐hydroxybutyrates were developed and subsequently applied to investigate Saiga horn extract for the presence of these compounds, which might explain its biological actions, particularly for its antipyretic and procoagulant properties.     

A novel mass spectrometry‐based method for simultaneous determination of asymmetric and symmetric dimethylarginine,l‐arginine and l‐citrulline optimized for LC‐MS‐TOF and LC‐MS/MS

Nitric oxide (NO) is a regulatory molecule involved in many biological processes. NO is produced by nitric oxide synthase by conversion of l‐ arginine to l‐ citrulline. l‐ Arginine methylated derivatives, asymmetric and symmetric dimethylarginines (asymmetric dimethylarginine, ADMA, and symmetric dimethylarginine, SDMA), regulate l‐ arginine availability and the activity of nitric oxide synthase. As such, they have been frequently investigated as potential biomarkers in pathologies associated with dysfunctions in NO synthesis. Here, we present a new multistep analytical methodology based on liquid chromatography combined with mass spectrometry for the accurate identification of l‐ arginine, l‐ citrulline, ADMA and SDMA. Compounds are measured as stable 2,3,4,5,6‐pentafluorobenzoyl chloride derivatives, which allows for simultaneous analysis of all compounds through chromatographic separation of ADMA and SDMA using a reverse‐phase column. Serum aliquots (100 μL) were spiked with isotope‐labeled internal standards and sodium carbonate buffer. The derivatization process was carried out at 25°C for 10 minu using pentafluorobenzoyl chloride as derivatization reagent. Calibration demonstrated good linearity (R ² = 0.9966–0.9986) for all derivatized compounds. Good accuracy (94.67–99.91%) and precision (1.92–11.8%) were observed for the quality control samples. The applicability of the method was evaluated in a cohort of angiological patients and healthy volunteers. The method discerned significantly lower l‐ arginine and l‐ citrulline in angiologic patients. This robust and fast LC‐ESI‐MS method may be a useful tool in quantitative analysis of l‐ arginine, ADMA, SDMA and l‐ citrulline.     

Multiresidue analysis of nine β‐agonists in animal muscles by LC‐MS/MS based on a new polymer cartridge for sample cleanup

A novel, sensitive, and reliable LC‐MS/MS method for multiresidue analysis of nine β‐agonists (salbutamol, terbutaline, cimaterol, fenoterol, clorprenaline, ractopamine, tulobuterol, clenbuterol, and penbuterol) in four farm animal muscles was developed. Muscle matrix was extracted with acetonitrile–10% sodium carbonate solution, and then was subjected to cleanup using a SPE cartridge packed with new polymer synthesized in acetone. Chromatographic separation of the components was performed on a Luna C₁₈ column using 0.1% of formic acid in water and acetonitrile. The mass spectrometer was operated in the positive electrospray mode. Good precision and accuracy were obtained for all analytes (except for fenoterol) at the spiked three levels of 1.0, 10, and 50 μg/kg. The decision limit and detection capability of nine β‐agonists ranged from 0.04 to 0.18 and 0.15 to 0.69 μg/kg, respectively. The method developed was successfully applied to the monitoring of nine β‐agonists in pork, beef, mutton, and chicken from Chinese markets.     

Multiplexed therapeutic drug monitoring of antipsychotics in dried plasma spots by LC‐MS/MS

In this work, a convenient method for the therapeutic monitoring of seven common antipsychotic drugs in “dried plasma spot” samples has been developed. It is based on the liquid chromatography tandem mass spectrometry technique, operating in multiple reaction monitoring mode, and a straightforward procedure for the simultaneous extraction of all antipsychotics in a single step, with high extraction yield. The method was fully validated with proper accuracy, precision, selectivity and sensitivity, for all the drugs. Limits of quantification were 0.12, 1.09, 1.46, 1.47, 5.70, 1.32, 1.33 µg/L for haloperidol, aripiprazole, olanzapine, quetiapine, clozapine, risperidone, and paliperidone, respectively. Accuracy, intra‐ and interday precision values were <10% for all drugs at all concentration levels examined. The method was tested in the analysis of 30 plasma samples from real patients for each drug. The proposed analytical approach, by combining practical and logistical advantages of microsampling with liquid chromatography tandem mass spectrometry analytical performance, could offer an ideal strategy for accurate and timely therapeutic drug monitoring of antipsychotic drugs in most clinical settings, even in remote centers and/or in out‐patient settings, bringing so many potential improvements in psychiatric patient care.     

复合免疫亲和柱-高效液相色谱法同时测定谷物及其制品中9种真菌毒素

建立了复合免疫亲和柱-在线光化学衍生-高效液相色谱同时测定谷物及其制品中9种真菌毒素的检测方法。以乙腈-水（80：20,v/v）混合溶液提取样品中9种真菌毒素,提取液经自制真菌毒素复合免疫亲和柱净化,采用高效液相色谱进行分离,在线光化学衍生后进入荧光检测器测定,外标法定量。结果表明,9种真菌毒素在相应浓度范围内线性关系良好,相关系数均大于0.999;在低、中、高3个不同加标浓度下,9种真菌毒素的回收率均大于80%,相对标准偏差（RSD）为1.0%~5.6%;方法的检出限（LOD）为0.02~5.00 μg/kg,定量限（LOQ）为0.07~16.70 μg/kg。该方法具有重现性好、灵敏度高、结果准确的特点,适用于谷物及其制品中9种真菌毒素残留的分析检测。     

LC‐MS analysis of estradiol in human serum and endometrial tissue: Comparison of electrospray ionization,atmospheric pressure chemical ionization and atmospheric pressure photoionization

Accurate measurement of estradiol (E2) is important in clinical diagnostics and research. High sensitivity methods are critical for specimens with E2 concentrations at low picomolar levels, such as serum of men, postmenopausal women and children. Achieving the required assay performance with LC–MS is challenging due to the non‐polar structure and low proton affinity of E2. Previous studies suggest that ionization has a major role for the performance of E2 measurement, but comparisons of different ionization techniques for the analysis of clinical samples are not available. In this study, female serum and endometrium tissue samples were used to compare electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) in both polarities. APPI was found to have the most potential for E2 analysis, with a quantification limit of 1 fmol on‐column. APCI and ESI could be employed in negative polarity, although being slightly less sensitive than APPI. In the presence of biological background, ESI was found to be highly susceptible to ion suppression, while APCI and APPI were largely unaffected by the sample matrix. Irrespective of the ionization technique, background interferences were observed when using the multiple reaction monitoring transitions commonly employed for E2 (m/z 271 > 159; m/z 255 > 145). These unidentified interferences were most severe in serum samples, varied in intensity between ionization techniques and required efficient chromatographic separation in order to achieve specificity for E2. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid analysis of constituents of Radix Cyathulae using hydrophilic interaction‐reverse phase LC‐MS

A hydrophilic interaction chromatography (HILIC) and reverse‐phase liquid chromatography (RPLC) coupled with electrospray TOF MS method was developed for the analysis and characterization of constituents in the radix of Cyathula officinalis Kuan. Separation parameters of HILIC such as buffer pH, mobile phase strength, and organic modifier were evaluated. Fructose, glucose, and sucrose were identified by HILIC‐ESI/TOF MS. Reverse‐phase liquid chromatography‐ESI/TOF MS were applied for quick and sensitive identification of major saponins in Cyathula officinalis. In‐source collision‐induced dissociation has been performed to elucidate the fragmentation pathways of oleanane‐, hederagenin‐, and gypsogmin‐type saponins. Twelve saponins were characterized in this plant for the first time, and four of them were presumed to be new compounds. In addition, one phytoecdysteroid (cyasterone) and one coumarin (6,7‐dimethoxycoumarin) were detected at the same time. The present method was capable of rapid characterizing and providing structure information of constituents from herbal drugs.     

Magnetic solid‐phase extraction based on carbon nanotubes for the determination of polyether antibiotic and s‐triazine drug residues in animal food with LC–MS/MS

Carbon nanotubes‐magnetic nanoparticles, comprising ferroferric oxide nanoparticles and carbon nanotubes, were prepared through a simple one‐step synthesis method and subsequently applied to magnetic solid‐phase extraction for the determination of polyether antibiotic and s‐triazine drug residues in animal food coupled with liquid chromatography with tandem mass spectrometry. The nanocomposites were characterized by transmission electron microscopy, X‐ray diffraction, and vibrating sample magnetometry. The components within the nanocomposites endowed the material with high extraction performance and manipulative convenience. Compared with carbon nanotubes, the as‐prepared carbon nanotubes‐magnetic nanoparticles showed better extraction and separation efficiencies for polyether antibiotics and s‐triazine drugs thanks to the contribution of the iron‐containing magnetic nanoparticles. Various experimental parameters affecting the extraction efficiency had been investigated in detail. Under the optimal conditions, the good linearity ranging from 1 to 200 μg/kg for diclazuril, toltrazuril, toltrazuril sulfone, lasalocid, monensin, salinomycin, narasin, nanchangmycin, and maduramicin, low limits of detection ranging from 1 to 5 μg/kg, and satisfactory spiked recoveries (77.1–91.2%, with the inter relative standard deviation values from 4.0 to 12.2%) were shown. It was confirmed that this novel method was an efficient pretreatment and enrichment procedure and could be successfully applied for extraction and determination of polyether and s‐triazine drug residues in complex matrices.     

Structural identification of degradants of moexipril by LC–MS/MS

A gradient LC–MS method was developed for the identification and characterization of degradants of moexipril using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS). Moexipril was subjected to hydrolysis (acid, base and neutral), oxidation, photolytic and thermal degradation conditions as mentioned in ICH guidelines Q1A (R2). The drug degraded under hydrolysis, oxidation and photolytic conditions, but it was stable under thermal conditions. In total, five degradants were formed and separated on an Agilent XDB C‐18 column (4.6 × 150 mm, 5 μm) in a gradient elution method. Four degradants ( D1 , D2 , D4 and D5 ) under acidic conditions, three degradants ( D2 , D3 and D4 ) under basic conditions and three degradants ( D1 , D4 and D5 ) under neutral and oxidative stress conditions were formed. In addition, two degradants ( D4 and D5 ) were formed under photolytic stress conditions. To elucidate the structures of degradants, fragmentation of moexipril and its degradants was studied using LC–MS/MS experiments and accurate mass measurements (HRMS) data. The fragment ions in the product ion tandem mass spectra of all the degradants were compared with those of moexipril and assigned the probable structures for the degradants.     

A robust LC–MS/MS assay with online cleanup for measurement of serum testosterone

Accurate measurement of low levels of testosterone is critical for diagnosis and treatment of androgen disorders. The very low concentrations of testosterone in children, females, and males with androgen suppression therapies necessitate the use of mass spectrometry‐based methods. We aimed to develop a liquid chromatography with tandem mass spectrometry method with simplified sample preparation and online solid‐phase extraction cleanup to achieve enhanced precision, accuracy, robustness, and cost‐effectiveness. The assay was linear from 10 to 20 000 pg/mL with an analytical recovery of 93–104%. The total coefficient of variation was 2.5, 1.9, and 1.7% at concentration levels of 348, 5432, and 10 848 pg/mL, respectively. No significant carryover was observed from samples with concentrations up to 20 000 pg/mL. No significant interference was observed from androstenedione, dehydroepiandrosterone, epi‐testosterone, and estriol. Comparison with CDC Hormone Standardization program (HoSt) reference samples with defined values (n = 40) showed a Deming regression slope of 0.963, intercept of 28.06 pg/mL, standard error of estimate was 66.9, a correlation coefficient of 0.9996, and a mean bias of ?0.6%. The method met the accuracy criteria by the CDC HoSt program. In addition, we achieved >12 000 injections on a single analytical column without significant performance deterioration due to the specific online solid‐phase extraction settings.     

Determination of penicillins in milk using LC‐UV,LC‐MS and LC‐MS/MS

The aim of this work is to establish a method for the simultaneous determination of eight penicillins in milk samples by LC‐UV, LC‐MS and LC‐MS/MS. The procedure involves a step for clean‐up and to preconcentrate the analytes by SPE and a subsequent chromatographic analysis. LC‐UV, LC‐MS and LC‐MS/MS have been used for the simultaneous quantification of penicillins in milk. The proposed methods have been validated according to the EU guideline and present LOQ below the maximum limits of residues (MRLs) established by the European Union for penicillins in milk. The developed methods were applied to different milk samples obtained from cows medicated with penicillins.