Single-laboratory validation of a modified liquid chromatographic method with UV detection for determination of trenbolone residues in bovine liver and muscle

Trenbolone acetate is a synthetic testosterone analog registered for use in a number of countries as a growth-promoting hormone, applied as an implant in the ears of feedlot cattle. The method is intended for the detection and quantitation of trace amounts of alpha- and beta-trenbolone in bovine tissues (muscle, liver) by liquid chromatography (LC) with UV detection and eliminates the use of the structural analog, 19-nortestosterone, as an internal standard. Trenbolone residues are extracted from tissues that have been homogenized in sodium acetate with a 3-phase liquid-liquid extraction by adding a mixture of water-acetonitrile-dichloromethanehexane, with trenbolone residues preferentially partitioned into the middle acetonitrile layer. The extract is passed through solid-phase extraction cartridges (both C18 and silica gel) using, respectively, methanol-water and acetone-toluene as eluents. Reversed-phase high-performance LC separation is performed, an octadecyl-bonded column with methanol-acetonitrile-water used as mobile phase for sample analysis. The limit of detection is 0.2 ng/g in muscle tissue and 0.6 ng/g in liver tissue, with coefficients of variation of 3.5-12.1% for alpha- and beta-trenbolone at concentrations from 0.2 to 4.0 ng/g fortified in muscle and 3.3-26.0% from liver fortified at 0.6-10.0 ng/g. Absolute recoveries of 40-130% were observed, but the use of fortified matrix curves eliminated recovery correction. Critical control points were identified in a pH adjustment step and an evaporation step during method validation, which included ruggedness testing. Analysis of incurred tissues (bovine liver and muscle) stored at -20 degrees C for over 25 weeks did not identify any significant loss of residues.     

Distribution of trenbolone residues in liver and various muscle groups of heifers that received multiple implants at the recommended site of application

Twenty heifers which were each administered 3 or 4 implants containing trenbolone acetate were slaughtered at 30 days post-implantation. Liquid chromatographic analyses were conducted on muscle collected from the rump, loin, shoulder, and neck, and on the liver of each animal. Residues present in liver were primarily 17alpha-trenbolone, and the residues found in the various muscle samples were primarily 17beta-trenbolone. The mean concentration of 17alpha-trenbolone in liver was 4.3 +/- 2.3 ng/g; the mean concentration of 17beta-trenbolone in muscle tissues was < 0.4 ng/g. There was a small but statistically significant effect of the number of implants used on the mean concentration of residues in loin muscles; animals with 3 trenbolone implants had higher mean residue concentrations than animals with 4 trenbolone implants. This suggests that, though the impact of implant numbers on the mean concentration of residues in muscle tissues is negligible relative to currently generally accepted maximum residue levels, mechanisms may exist for selective distribution and retention of residues within different muscle groups.     

Multiresidue determination of zeranol and related compounds in bovine muscle by gas chromatography/mass spectrometry with immunoaffinity cleanup

A gas chromatography/mass spectrometry (GC/MS) method with immunoaffinity cleanup was developed for the determination of zeranol and related compounds, taleranol, zearalanone, and alpha-zearalenol in bovine muscle. Muscle samples were extracted with methanol and cleaned up with immunoaffinity chromatography (IAC) columns containing monoclonal antibodies raised against zeranol coupled to CNBr-activated Sepharose 4B. After derivatization, the compounds were analyzed by GC/MS. The dynamic column capacities for zeranol, taleranol, zearalanone, and alpha-zearalenol were 2639.7, 2840.3, 2731.5, and 2736.3 ng/mL Sepharose gel, respectively. The limits of detection and quantification were 0.5 and 1.0 ng/g, respectively, for all 4 compounds. Mean recoveries were 79.6-110.7% with coefficients of variation of 3.2-11.4% at spiked levels of 1.0-5.0 ng/g. This IAC-GC/MS method may be used for the determination of zeranol, taleranol, zearalanone, and alpha-zearalenol residues in bovine muscle, and possibly other tissues.     

Validation study for the determination of tetracycline residues in animal tissues.

An interlaboratory study of the liquid chromatographic (LC) determination of tetracyclines--oxytetracycline (OTC), tetracycline (TTC), and chlortetracycline (CTC)--in animal tissues was conducted. Isolation was performed with oxalic buffer followed by dechelation and deproteination with oxalic acid-acetonitrile. The extract was cleaned with a styrene-divinylbenzene cartridge. LC analysis was performed with a PLRP-S column and 0.01 M oxalic acid-acetonitrile (75 + 25, v/v) as mobile phase. Participants analyzed 2 control and 10 fortified porcine muscle and kidney samples. Additionally, porcine muscle samples containing incurred residues of tetracyclines were analyzed. Mean recoveries of fortified residues from porcine tissue ranged from 76.00 to 86.89%. Repeatabilities varied from 2.05% for OTC to 3.61% for TTC for muscle samples and from 6.75% for CTC to 8.74% for OTC for kidney samples. Reproducibilities ranged from 2.05 to 4.30% for muscle samples and from 15.77 to 18.81% for kidney samples.     

Simultaneous determination of quinolone antibacterials in bovine milk by liquid chromatography-mass spectrometry

A new liquid chromatography-mass spectrometry (LC-MS) method has been developed and validated for the simultaneous determination of eight quinolone antibacterials for veterinary use in processed bovine milk samples. The quinolones studied included marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid and flumequine. Also, a new sample-treatment procedure was used for extraction and preconcentration of these compounds. It involved defatting by centrifugation, protein precipitation by adding a mixture of glacial acetic acid-acetonitrile and removing acetonitrile with dichloromethane; finally, the acidified aqueous layer was evaporated to dryness in a speed vac system, resuspended in the mobile phase and filtered prior to LC injection. The mobile phase was composed of a formic acid aqueous solution 0.1% (v/v) and acetonitrile, with an initial composition of water-acetonitrile 95: 5 (v/v) and using linear gradient elution. Norfloxacin was used as internal standard. The limits of quantification found (2-7 ng g(-1)) were in all cases lower than the maximum residue limits tolerated by the European Union for these compounds in milk.     

Micro-Plate Chemiluminescence Enzyme Immunoassay for Determination of Zeranol in Bovine Milk and Urine

Zeranol (α-Zearalanol, α-ZAL), well-known as an anabolic promoter, was officially banned in Europe due to its potential carcinogenic and endocrine-disrupting biological activities. In this study, a method for the determination of zeranol residues in bovine milk and urine was developed based on micro-plate chemiluminescence enzyme immunoassay (CLEIA). The limit of detection (LOD) was 50 ng/L, and the linear range was between 100 ng/L and 4510 ng/L, with a correlation coefficient (R²)0.9982. The recoveries of zeranol in bovine milk and urine were between 84.7% and 123.6%. This study showed that CLEIA was a reliable, convenient, and sensitive method for screening zeranol residues in bovine milk and urine.     

Determination of afloqualone in human plasma using liquid chromatography/tandem mass spectrometry: Application to pharmacokinetic studies in humans

Two methods for determining the central-acting muscle relaxant afloqualone in human plasma were developed and compared using API2000 and API4000 liquid chromatography tandem mass spectrometry (LC/MS/MS) systems. In the API2000 LC/MS/MS system, afloqualone and the internal standard methaqualone were extracted from plasma using a methyl-tertiary ether. After drying the organic layer, the residue was reconstituted in a mobile phase (0.1% formic acid-acetonitrile:0.1% formic acid buffer, 80:20 v/v) and injected onto a reversed-phase C₁₈ column. The isocratic mobile phase was eluted at 0.2 ml/min. The ion transitions monitored in multiple reaction-monitoring mode were m/z 284 → 146 and 251 → 117 for afloqualone and methaqualone, respectively.Sample preparation for the API4000 LC/MS/MS system involved simple protein precipitation with an organic mixture (methanol:10% ZnSO₄ = 8:2). The ion transitions monitored in multiple reaction-monitoring mode were m/z 284 → 146 and 251 → 131 for afloqualone and methaqualone, respectively.In both assays, the coefficient of variation of the precision was less than 11.8%, the accuracy exceeded 91.5%, the limit of quantification was 0.5 ng/ml, and the limit of detection was 0.1 ng/ml for afloqualone. Two methods were used to measure the plasma afloqualone concentration in healthy subjects after a single oral 20-mg dose of afloqualone. During subsequent application of the methods, we observed that high-concentration plasma samples (>7 ng/ml) prepared using the protein precipitation method resulted in about 20% higher afloqualone concentrations than with plasma samples prepared using the liquid-liquid extraction method. We believe that this phenomenon was related to the cleanness of the sample and its chemical nature.     

Determination of chloramphenicol residues in milk, eggs, and tissues by liquid chromatography/mass spectrometry

A new liquid chromatography/mass spectrometry (LC/MS) method is presented for the determination of chloramphenicol (CAP) residues in milk, eggs, chicken muscle and liver, and beef muscle and kidney. CAP is extracted from the samples with acetonitrile and defatted with hexane. The acetonitrile extracts are then evaporated, and residues are reconstituted in 10mM ammonium acetate--acetonitrile mobile phase and injected into the LC system. CAP is determined by reversed-phase chromatography using an Inertsil ODS-2 column and MS detection with negative ion electrospray ionization. Calibration curves were linear between 0.5-5.0 ng/g for all matrixes studied. The relative standard deviations for measurements by this method were generally <12%, and average recoveries ranged from 80 to 120%, depending on the matrix involved. The method detection limits of CAP ranged from 0.2 to 0.6 ng/g, which are comparable to previously reported results. The proposed method is rapid, simple, and specific, allowing a single analyst to easily prepare over 40 samples in a regular working day.     

Determination of chlorpyrifos and its metabolites in rat brain tissue using coupled-column liquid chromatography/electrospray ionization tandem mass spectrometry

A method has been developed to quantify chlorpyrifos (O,O-diethyl-O-[3,5,6,-trichloro-2-pyridyl] phosphorothionate) and its metabolites chlorpyrifos-oxon (O,O-diethyl-O-[3,5,6,trichloro-2-pyridinyl] phosphate) and TCP (3,5,6,-trichloro-2-pyridinol) in rat brain tissue by coupled-column liquid chromatography/electrospray ionization tandem mass spectrometry (LC/LC/ESI-MS/MS). Rat brains were homogenized and treated by protein precipitation using ice-cold acetonitrile. The supernatant was directly injected onto the coupled-column system. Sample clean-up was achieved on a Zorbax Extend-C(18) column (2.1 x 50 mm, 5 microm) using a mobile phase of acetonitrile/water with 0.0025% formic acid (40:60, v/v). The compounds were separated isocratically on a Zorbax Eclipse XDB C(8) column (2.0 x 150 mm, 5 microm) using a mobile phase of acetonitrile/water with 0.0025% formic acid (75:25, v/v). Chlorpyrifos and chlorpyrifos-oxon were detected in positive ion mode using multiple reaction monitoring (MRM). TCP was detected in negative ion mode using precursor-to-precursor transition monitoring. The method was validated and the specificity, linearity, limit of quantitation (LOQ), precision, accuracy, stability, and recoveries were determined. Calibration curves for all three analytes yielded correlation coefficients of 0.993 or greater. The LOQs were 25.3 ng/g for chlorpyrifos and 6.3 ng/g for chlorpyrifos-oxon and TCP. All precision relative standard deviations (RSDs) were less than 16% for the LOQ and less than 11% for the other QC samples. This method was successfully applied to six rats that were injected subcutaneously with chlorpyrifos.     

Determination of cylindrospermopsin in freshwaters and fish tissue by liquid chromatography coupled to electrospray ion trap mass spectrometry

Cylindrospermopsin (CYN) is a toxic alkaloid‐like compound produced by some strains of cyanobacteria, procariotic organisms occurring in water blooms, observed worldwide in eutrophic lakes and drinking water reservoirs. Methods for determination of CYN in freshwater and fish muscle by liquid chromatography coupled to electrospray ion trap mass spectrometry are herein described. The performances of both methods are reported; ion trap LC/ESI‐MS/MS resulted highly selective and reliable in unambiguous identification of CYN, based on monitoring the precursor ion and three product ions. The methods developed showed satisfactory mean recoveries (higher than 63.6%) and relative standard deviations, ranging from 5.8 to 9.8%. The limits of quantification at 0.10 ng/mL in freshwaters and 1.0 ng/g in fish muscle, respectively, allow for determination of CYN also in early contamination stages. Ion trap LC/ESI‐MS/MS was successfully applied to the identification and quantification of CYN in water and cyanobacteria extracts from Lake Averno, near Naples, representing the first case of contamination described in southern Italy. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of residues of dipyrone and its major metabolites in milk, bovine muscle, and porcine muscle by liquid chromatography/mass spectrometry

A new liquid chromatography/mass spectrometry (LC/MS) method for the determination of dipyrone (DIP) and the DIP-related residues 4-formylaminoantipyrine (FAA), 4-aminoantipyrine (AA), and 4-methylaminoantipyrine (MAA) in milk, bovine muscle, and porcine muscle is presented. The analytes are extracted from the sample with methanol and defatted with hexane. The methanol extracts are then concentrated and injected into the LC system. Compounds are determined by reversed-phase LC using an Inertsil ODS-3 column with ammonium formate-acetonitrile mobile phase and MS detection using positive-ion electrospray ionization. Calibration curves were linear between 0.02 and 0.20 microg/g matrix equivalent concentration for FAA, AA, and MAA, and between 0.2 and 2.0 microg/g for DIP. The relative standard deviations for measurements by the proposed method were <11% for milk and porcine samples, with slightly greater variability for bovine samples. Average recoveries ranged from 82 to 128%, depending on the compound and matrix involved. The method detection limits of FAA, AA, and MAA were <0.02 microg/g for all matrixes tested, while those of DIP were <0.13 microg/g. The proposed method is rapid, simple, and specific, allowing a single analyst to easily prepare over 20 samples in a regular working day.     

Thin-layer chromatographic detection of zeranol and estradiol in fortified plasma and tissue extracts with Fast Corinth V.

In an attempt to improve sensitivity of thin-layer chromatographic (TLC) analysis and selectivity of visualizing agents for detection of estrogenic anabolic hormones, several dyes were screened for their chromogenic interactions with estrone, estradiol, diethylstilbestrol (DES), zeranol (zearalanol), zearalanone, and mycotoxins, zearalenone and zearalenol. Fast Corinth V salt was selected for its relatively high sensitivity. These anabolic compounds were separated by TLC and visualized with Corinth V and the results compared to iodine and starch visualization. Fortified bovine plasma and tissues (kidney, liver and muscle) and chicken muscles were analyzed after a clean-up procedure using solid-phase dual columns of alumina and anion-exchange resin. Iodine-starch clearly detected 4 ng of estradiol and DES while zeranol and zearalenone were detected at higher levels (10 ng). Fast Corinth V showed distinct spots with 2 ng of zeranol and 4 ng of zearalenone while faint spots were observed with estradiol and estrone standards. DES was not detectable at these levels. Less background interference was observed with Corinth V than with iodine-starch. The former confirmed spots detected by iodine-starch. This study suggests its selectivity for detection of zeranol and its metabolite, zearalanone, in the presence of steroidal compounds.     

Determination of type B fumonisin mycotoxins in maize and maize-based products by liquid chromatography/tandem mass spectrometry using a QqQlinear ion trap mass spectrometer

A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for determining the type B fumonisin mycotoxins in corn-based foodstuffs is described. Fumonisins FB1 and FB2 were extracted from a 1 g sample by homogenization with acetonitrile/water (75:25, v/v, 50 mmol/L formic acid, 25 mL final volume) and the extract was defatted on C18 phase. Volumes of 5 mL of crude extracts were cleaned up on Carbograph-4 cartridges. The final solution was analyzed by HPLC with electrospray ionization mass spectrometry in positive ion mode using multiple reaction monitoring with a QqQ linear ion trap mass spectrometer. Recoveries for spiked corn-based foodstuffs ranged from 91-105% (RSD% < or =8%), and method detection limits were < or =2 ng/g for FB1 and < or =1 ng/g for FB2. Two different spiking levels were tested (5000 and 100 ng/g for FB1, 1000 and 20 ng/g for FB2). Quantitation was achieved by an external calibration procedure using matrix-matched standards, with diclofenac added post-cleanup as internal standard for the LC/MS/MS analyses. Calibration curves showed linearity in the concentration range 0.005-5 ng/microL of final extract (0.992 < or = R2< or =0.995). Two other fumonisins, FB3 and FB4, were identified in naturally contaminated samples of corn meal using an information-dependent acquisition protocol that looped three experiments, including neutral loss scan, enhanced resolution scan, and enhanced product ion scan. FB3 and FB4 quantitation was estimated as peak area ratios relative to the FB2 response in view of the lack of both standards. This work also includes an application of the present LC/MS/MS method to some maize and maize-based product samples (corn meal, cornflakes and popcorn) collected from Italian stores. FB1 and FB2 contamination levels exceeding the European Union recommendation were found in 8 out of 15 corn meal samples.     

Simple confirmatory assay for analyzing residues of aminoglycoside antibiotics in bovine milk: hot water extraction followed by liquid chromatography-tandem mass spectrometry

A simple, selective and sensitive procedure for determining nine widely used aminoglycoside antibiotics (AGs) in bovine whole milk is presented. It is based on matrix solid-phase dispersion with heated water, at 70 degrees C, as extractant followed by liquid chromatography (LC)-tandem mass spectrometry (MS) using an electrospray ion source. After acidification and filtration, 0.2 ml of the aqueous extract was injected into the LC column. MS data acquisition was performed in the multi reaction monitoring mode, selecting two (three, when possible) precursor ion > product ion transitions for each target compound. Analyte recoveries ranged between 70 and 92%. Using aminosidine (an AG not used in veterinary medicine) as surrogate internal standard, the accuracy of the method at three spike levels varied between 80 and 107% with R.S.D. not larger than 11%. The limits of quantification were between 2 ng/ml (apramycin) and 13 ng/ml (streptomycin). They are well below the tolerance levels set by both the European Union and the U.S. Food and Drug Administration.     

Development of bovine muscle, liver and urine reference materials for zeranol – preparation, homogeneity and stability

 Samples of bovine muscle, liver and urine, zeranol-free (RM 508, RM 509 and RM 510, respectively) and zeranol-containing (RM 511, RM 512 and RM 513, respectively) were prepared and tested as candidate reference materials. Preliminary studies on achievement of target zeranol content, intercomparison of analytical methods (HPLC-RIA and GC-MS) and effects of lyophilisation and irradiation on zeranol content are described. The preparation of the materials and testing for homogeneity and stability of zeranol in the materials are discussed. The coefficients of variation for zeranol determinations for between-vial homogeneity (4.0%, 4.4% and 4.6% for muscle, liver and urine, respectively) are similar to those for the analytical method (4.0%, 7.3% and 6.8% for muscle, liver and urine, respectively) indicating that the materials are homogeneous. Stability data over a 12-month storage period at temperatures ranging from −18 °C to +37 °C indicate that the materials are sufficiently stable for use as reference materials. This paper is dedicated to the memory of Dr Hilary Stevenson (The Queen’s University of Belfast/DANI) who, together with Mr. W. Graham, facilitated the irradiation of the lyophilised materials.--> Received: 18 June 1996/Revised: 19 August 1996/Accepted: 23 August 1996     

Rapid determination of nosiheptide in meat and egg by liquid chromatography with fluorescence detection

A procedure was developed to determine nosiheptide residues in marketed meat and egg. Acetonitrile was used for the extraction, and the extract was partitioned with hexane to remove fat. The lower layer was reconstructed and quantitated by liquid chromatography using fluorescence detection at 357 nm excitation and 500 nm emission. The mobile phase consisted of 0.025% phosphoric acid-acetonitrile (50 + 50, v/v). Recoveries of nosiheptide from fortified samples ranged from 91.3 to 95.2% for swine muscle, 88.6 to 92.7% for chicken muscle, and 86.3 to 86.8% for egg. The method was used to monitor swine and chicken muscle and egg (20 samples each) in the market. Nosiheptide was not determined in all 60 samples.     

Determination of perfluorooctanoic acid and perfluorooctanesulfonate in human tissues by liquid chromatography/single quadrupole mass spectrometry

A method is described that permits the measurement of the levels of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) in human liver, kidney, adipose tissue, brain, basal ganglia, hypophysis, thyroid, gonads, pancreas, lung, skeletal muscle and blood, even in subjects not occupationally exposed to these compounds. The purification of samples involved the use of trifunctional (tC18) and strong anion-exchange (SAX) solid-phase extraction cartridges, and the analysis utilized a high-performance liquid chromatograph coupled to a single quadrupole mass spectrometer (LC/MS). The analyses were conducted on a mixed-bed reversed-phase column by gradient runs using 3 mM ammonium acetate/methanol mixtures at different proportions as the mobile phase. The detector was used in electrospray negative ion mode by recording simultaneously the ions m/z 413.0 (PFOA) and 499.0 (PFOS). Perfluorononanoic acid (PFNA), added to the samples before the purification, was used as the internal standard (ion monitored = m/z 463.6). The recovery rates of the extraction procedure ranged from 79.6 to 95.6% (CV% 1.7-7.4%) for PFOA, from 79.7 to 100.8% (CV% = 1.2-7.1) for PFOS, and from 89.1 to 102.3% (CV% = 0.9-5.2 %) for PFNA. The calibration curves were linear up to at least 400 ng of analytes per gram of tissue. The detection limits (signal-to-noise ratio = 3) were 0.1 ng/g for both PFOA and PFOS measured in all tissues except adipose tissue, where the limits were about 0.2 ng/g. The content of analytes in tissues varied from 0.3 to 3.8 ng/g (respectively: basal ganglia and lung) for PFOA, and from 1.0 to 13.6 ng/g (respectively: skeletal muscle and liver) for the linear isomer of PFOS. The method is suitable to evaluate the content of PFOA and PFOS in different tissues taken from the general population exposed to very low concentrations of these pollutants.     

Quantitative determination of marine toxins associated with diarrhetic shellfish poisoning by liquid chromatography coupled with mass spectrometry

Quantitative determination by liquid chromatography (LC) coupled with mass spectrometry (MS) was achieved for the following 10 toxins found in association with diarrhetic shellfish poisoning: okadaic acid (OA), dinophysistoxin-1 (DTX1), 7-O-palmitoylokadaic acid (palOA), 7-O-palmitoyldinophysistoxin-1 (pa1DTX1), pectenotoxin-1 (PTX1), pectenotoxin-2 (PTX2), pectenotoxin-2 seco acid (PTX2SA), pectenotoxin-6 (PTX6), yessotoxin (YTX), and 45-hydroxyyessotoxin (YTXOH). Toxins in 2 g of the adductor muscle or the digestive glands of scallops, Patinopecten yessoensis, were extracted with 18 ml of methanol-water (9:1, v/v), freed of polar contaminants by partition between chloroform and water, and treated by solid-phase extraction on a silica cartridge column. Samples containing YTXOH were purified separately on a buffered reversed-phase column. Chromatographic separation was achieved by the following combinations of columns and mobile phases: a Symmetry C18 column with acetonitrile-0.05% acetic acid (7:3, v/v) for OA, DTX1, PTX6 and PTX2SA; a Develosil ODS column with the same mobile phase for PTX1 and PTX2; a Capcellpak column with methanol-2.5% acetic acid (98:2, v/v) for palOA and palDTX1; and an Inertsil ODS column with methanol-0.2 M ammonium acetate (8:2, v/v) for YTX and YTXOH. Carboxylic acid toxins were selectively monitored on [M-H]- ions, sulfated toxins on [M-Na]-ions, and neutral toxins on [M+NH4]+ ions. Average recoveries of the toxins spiked to tissue homogenates ranged from 70 to 134%. Detection limits in the muscle ranged from 5 to 40 ng/g and those in the digestive glands from 10 to 80 ng/g.     

Determination of mazindol in human oral fluid by high performance liquid chromatography–electrospray ionization mass spectrometry

Brazil is one of the countries most affected by abuse of stimulant medications by professional drivers, especially fenproporex, amfepramone and mazindol. Even though their sale is banned, they can be found in illegal markets, such as those located on the country's borders. The use of oral fluid to monitor drug levels has many advantages over plasma and urine because it is noninvasive, easier to collect and more difficult to adulterate. The aim of this study was to develop and validate a sensitive and specific method to quantify mazindol in human oral fluid by liquid chromatography–mass spectrometry (LC‐MS). The LC system consisted of an LC‐MS system operated in selected ion monitoring mode. The mobile phase was composed of water at pH 4.0, acetonitrile and methanol (60:15:25 v/v/v) at a flow rate of 1.0 mL/min and propranolol was used as internal standard. Total running time was 10 min. The lower limit of quantification was 0.2 ng/mL and the method exhibited good linearity within the 0.2–20 ng/mL range (r = 0.9987). A rapid, specific, sensitive, linear, precise and accurate method was developed for determination of mazindol in human oral fluid according to European Medicines Agency guidelines, and is suitable for monitoring mazindol levels in oral fluid of professional drivers. Copyright © 2014 John Wiley & Sons, Ltd.