Determination of anabolic steroids in muscle tissue by liquid chromatography–tandem mass spectrometry

A specific and sensitive multi-method based on liquid chromatography–tandem mass spectrometry using atmospheric pressure chemical ionization (LC–APCI–MS/MS) has been developed for the determination of 20 anabolic steroids in muscle tissue (diethylstilbestrol, β-estradiol, ethynylestradiol, α/β-boldenone, α/β-nortestosterone, methyltestosterone, β-trenbolone, triamcinolone acetonide, dexamethasone, flumethasone, α/β-zearalenol, α/β-zearalanol, zearalenone, melengestrol acetate, megestrol acetate and medroxyprogesterone acetate). The procedure involved hydrolysis, extraction with tert-butyl methyl ether, defattening and final clean-up with solid phase extraction (SPE) on Oasis HLB and Amino cartridges. The analytes were analyzed by reversed-phase LC–MS/MS, in positive and negative multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for the unambiguous confirmation of the hormones. The method was validated at the validation level of 0.5 ng/g. The accuracy and precision of the method were satisfactory. The decision limits CCα ranged from 0.03 to 0.14 ng/g while the detection capabilities CCβ ranged from 0.05 to 0.24 ng/g. The developed method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in muscle tissue and can be used for residue control programs.     

Determination of evodiamine and rutecarpine in human serum by liquid chromatography–tandem mass spectrometry

Evodiamine and rutecarpine are two kinds of indole alkaloids contained in the fruit of Evodiae fructus, which have been shown to exhibit various bioactivities in humans. A liquid chromatography–tandem mass spectrometric method (LC–MS/MS) was developed for the determination of evodiamine and rutecarpine in human serum. The serum was extracted by solid-phase extraction (SPE) and analyzed using a C18 column and a mobile phase consisting of methanol–water (85:15) solution containing 5 mmol/L ammonium formate at a flow rate of 0.5 mL/min. The mass spectrometer was operated in positive mode, employing the extracted ion chromatogram (EIC) for detection and quantitation of evodiamine (m/z 288) and rutecarpine (m/z 304). Good linear relationships between the peak area and the concentration were obtained in the ranges of 5.2–1040 ng/mL and 10.2–1020 ng/mL, with correlation coefficients (r) of 0.999 and 0.998, for evodiamine and rutecarpine, respectively. The repeatabilities (RSD, n=6) of quantitation for evodiamine and rutecarpine were 2.18–4.00% and 2.99–5.67%, respectively, and the recovery ranged from 90.5% to 98.1%. A comparative study of the different ionization and quantitation modes, including ESI–MS, ESI–MS/MS, APCI–MS and APCI–MS/MS, was also accomplished. The MS/MS fragmentation mechanism of the base peak ([M+H]⁺, m/z 304) of evodiamine was investigated in order to identify the analytes in more complicated body fluid samples.      

Determination of tandospirone in human plasma by a liquid chromatography–tandem mass spectrometry method

A rapid, sensitive, and selective liquid chromatography–tandem mass spectrometry method for the detection of tandospirone in human plasma is described. It was employed in a pharmacokinetic study. The analyte and internal standard diphenhydramine were extracted from plasma using liquid–liquid extraction, then separated on a Zorbax XDB C₁₈ column using a mobile phase of methanol–water–formic acid (80:20:0.5, v/v/v). The detection was performed with a tandem mass spectrometer equipped with an electrospray ionization source. Linearity was established in the concentration range of 10.0-5,000 pg/ml. The lower limit of quantification was 10.0 pg/ml. The intraday and interday relative standard deviation across three validation runs over the entire concentration range was less than 13%. Accuracy determined at three concentrations (25.0, 200, and 4,000 pg/ml for tandospirone) ranged from 94.4 to 102.1%. Each plasma sample was chromatographed within 3.4 min. The method proved to be highly selective and suitable for bioequivalence evaluation of different formulations containing tandospirone and clinical pharmacokinetic investigation of tandospirone.     

Determination of cyclopiazonic acid in food and feeds by liquid chromatography–tandem mass spectrometry

A new, fast and efficient multiple reaction monitoring (MRM) high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for the determination of cyclopiazonic acid (CPA) in mixed feed, wheat, peanuts and rice is presented. The analytical methodology involves sample extraction with an alkaline methanol–water mixture, defatting with hexane and quantification using HPLC–MS/MS without further treatment of sample extracts. Reversed-phase liquid chromatography using a C18 stationary phase coupled to negative mode electrospray triple quadrupole tandem mass spectrometry was applied. The limit of detection was 5 μg/kg while the limit of quantification was 20 μg/kg in the matrices investigated. The detector response was found to be linear over the range 25–250 μg/kg in feed and 25–500 μg/kg in wheat, peanuts and rice. The mean overall recoveries (n = 18) of CPA varied from 79% to 114% in the range of concentrations studied over a period of 4 months. Mean recoveries (n = 3 or 6) of CPA in wheat, peanuts and rice varied from 70% to 111%, 77% to 116% and 69% to 92%, respectively. The method was successfully applied to the analysis of feed and rice samples artificially infected with the fungal strain Penicillium commune, where the toxin was found at different levels.     

Determination of 76 pharmaceutical drugs by liquid chromatography–tandem mass spectrometry in slaughterhouse wastewater

A multi-residue method for the analysis of 76 pharmaceutical agents of nine classes of drugs (tetracyclines, macrolides, fluoroquinolones, β-agonists, β-blockers, diuretics, sedatives, sulfonamides and chloramphenicol) in slaughterhouse wastewater and a receiving river is presented. After simultaneous extraction with an Oasis HLB solid-phase extraction (SPE) cartridge and further purification using an amino SPE cartridge, analytes were detected by liquid chromatography–electrospray ionization-tandem mass spectrometry in positive or negative ion mode. Standard addition was used for quantification to overcome unavoidable matrix effects during ESI-MS analysis. Recoveries for most analytes based on matrix-matched calibration in different test matrices were >60%. The method quantification limits of 76 pharmaceuticals were in the range 0.2–30 ng/L. Nineteen compounds of 76 drugs were found in raw and treated slaughterhouse wastewater from four main slaughterhouses in Beijing. Sulfanamides (sulfanilamide, sulfameter), fluoroquenones (ofloxacin, pefloxacin, norfloxacin, ciprofloxacin, enrofloxacin), tetracyclines (tetracycline, oxytetracycline) and macrolides (kitasamycin, tylosin, erythromycin) were most frequently detected, with the highest levels up to ∼3 μg/L in slaughterhouse wastewater and ∼1 μg/L in treated wastewater. Illicit drugs for animal feeding such as clenbuterol and diazepam were commonly detected in slaughterhouse wastewater. These analytes were also observed in a river receiving slaughterhouse wastewater, with a highest level of up to 0.2 μg/L.     

Determination of biocides in different environmental matrices by use of ultra-high-performance liquid chromatography–tandem mass spectrometry

A sensitive and robust method using solid-phase extraction and ultrasonic extraction for preconcentration followed by ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS–MS) has been developed for determination of 19 biocides: eight azole fungicides (climbazole, clotrimazole, ketoconazole, miconazole, fluconazole, itraconazole, thiabendazole, and carbendazim), two insect repellents (N,N-diethyl-3-methylbenzamide (DEET), and icaridin (also known as picaridin)), three isothiazolinone antifouling agents (1,2-benzisothiazolinone (BIT), 2-n-octyl-4-isothiazolinone (OIT), and 4,5-dichloro-2-n-octyl-isothiazolinone (DCOIT)), four paraben preservatives (methylparaben, ethylparaben, propylparaben, and butylparaben), and two disinfectants (triclosan and triclocarban) in surface water, wastewater, sediment, sludge, and soil. Recovery of the target compounds from surface water, influent, effluent, sediment, sludge, and soil was mostly in the range 70–120?%, with corresponding method quantification limits ranging from 0.01 to 0.31?ng?L^?1, 0.07 to 7.48?ng?L^?1, 0.01 to 3.90?ng?L^?1, 0.01 to 0.45?ng?g^?1, 0.01 to 6.37?ng?g^?1, and 0.01 to 0.73?ng?g^?1, respectively. Carbendazim, climbazole, clotrimazole, methylparaben, miconazole, triclocarban, and triclosan were detected at low ng?L^?1 (or ng?g^?1) levels in surface water, sediment, and sludge-amended soil. Fifteen target compounds were found in influent samples, at concentrations ranging between 0.4 (thiabendazole) and 372?ng?L^?1 (methylparaben). Fifteen target compounds were found in effluent samples, at concentrations ranging between 0.4 (thiabendazole) and 114?ng?L^?1 (carbendazim). Ten target compounds were found in dewatered sludge samples, at concentrations ranging between 1.1 (DEET) and 887?ng?g^?1 (triclocarban).     

Qualitative detection of desmopressin in plasma by liquid chromatography–tandem mass spectrometry

This work describes a liquid chromatography–electrospray tandem mass spectrometry method for detection of desmopressin in human plasma in the low femtomolar range. Desmopressin is a synthetic analogue of the antidiuretic hormone arginine vasopressin and it might be used by athletes as a masking agent in the framework of blood passport controls. Therefore, it was recently added by the World Anti-Doping Agency to the list of prohibited substances in sport as a masking agent. Mass spectrometry characterization of desmopressin was performed with a high-resolution Orbitrap-based mass spectrometer. Detection of the peptide in the biological matrix was achieved using a triple-quadrupole instrument with an electrospray ionization interface after protein precipitation, weak cation solid-phase extraction and high performance liquid chromatography separation with an octadecyl reverse-phase column. Identification of desmopressin was performed using three product ions, m/z 328.0, m/z 120.0, and m/z 214.0, from the parent ion, m/z 535.5. The extraction efficiency of the method at the limit of detection was estimated as 40% (n = 10), the ion suppression as 5% (n = 10), and the limit of detection was 50 pg/ml (signal-to-noise ratio greater than 3). The selectivity of the method was verified against several endogenous and synthetic desmopressin-related peptides. The performance and the applicability of the method were tested by analysis of clinical samples after administration of desmopressin via intravenous, oral, and intranasal routes. Only after intravenous administration could desmopressin be successfully detected.     

Determination of non-steroidal anti-inflammatory drugs and their metabolites in milk by liquid chromatography–tandem mass spectrometry

Non-steroidal anti-inflammatory drugs are widely used for treatment of animals. According to Council Directive 96/23/EC, residues of these drugs must be monitored because of the potential risk they pose to the consumers' health. For this reason an LC-MS-MS method was developed for detection of wide range of NSAIDs, including both "acidic" NSAIDs (carprofen, diclofenac, flunixin, meloxicam, phenylbutazone, oxyphenbutazone, tolfenamic acid, mefenamic acid, naproxen, ketoprofen, ibuprofen, firocoxib, rofecoxib, and celecoxib) and "basic" NSAIDs (four metamizole metabolites). Analytes were extracted from milk samples with acetonitrile in the presence of ammonium acetate. One portion of the extract was directly analyzed for the presence of metamizole metabolites; a second portion was cleaned with an amino cartridge. All NSAIDs were separated on a Phenomenex Luna C8(2) column and analyzed by LC-MS-MS in negative (acidic NSAIDs) and positive (metamizole metabolites) ion modes. The method was validated in accordance with the requirements of Commission Decision 2002/657/EC. Within-laboratory reproducibility was in the range 7-28%, and accuracy was in the range 71-116%. The method enabled detection of all the analytes with the expected sensitivity, below the recommended concentrations. The method fulfills the criteria for confirmatory methods and, because of its efficiency, may also be used for screening purposes. The procedure was also successfully verified in the proficiency test organized by EU-RL in 2010. As far as the authors are aware, this is one of the first methods capable of detecting diclofenac residues below the MRL in milk (0.1 μg kg(-1)). An additional advantage is the possibility of simultaneous determination of "acidic" NSAIDs and metamizole metabolites.     

Determination of perfluorinated compounds in mollusks by matrix solid-phase dispersion and liquid chromatography–tandem mass spectrometry

Perfluorinated compounds (PFCs) have been used for over 40 years in different commercial and industrial applications mainly as surfactants and surface protectors and have become an important class of marine emerging pollutants. This study presents the development and validation of a new analytical method to determine the simultaneous presence of eight PFCs in different kinds of mollusks using matrix solid-phase dispersion (MSPD) followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Simplicity of the analytical procedure, low volume of solvent and quantity of sample required, low global price, and integration of extraction and clean-up into a single step, are the most important advantages of the developed methodology. Solvent, solid support (dispersing agent), clean-up sorbent, and their amounts were optimized by means of an experimental design. In the final method, 0.5 g of sample are dispersed with 0.2 g of diatomaceous earth and transferred into a polypropylene syringe containing 4 g of silica as clean-up sorbent. Then, analytes are eluted with 20 mL of acetonitrile. The extract is finally concentrated to a final volume of 0.5 mL in methanol, avoiding extract dryness in order to prevent evaporation losses and injected in the LC-MS/MS. The combination of this MSPD protocol with LC-MS/MS afforded detection limits from 0.05 to 0.3 ng g⁻¹. Also, a good linearity was established for the eight PFCs in the range from limit of quantification (LOQ) to 500 ng mL⁻¹ with R ² > 0.9917. The recovery of the method was studied with three types of spiked mollusk and was in the 64–126% range. Moreover, a mussel sample was spiked and aged for more than 1 month and analyzed by the developed method and a reference method, ion-pair extraction, for comparison, producing both methods statistically equal concentration values. The method was finally applied to the determination of PFCs in different kinds of mollusks revealing concentrations up to 8.3 ng g⁻¹ for perfluoroundecanoic acid.     

Analysis of amprolium by hydrophilic interaction liquid chromatography–tandem mass spectrometry

We present a fast liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the analysis of the coccidiostat amprolium in food samples. Tandem mass spectrometry in a triple quadrupole was used for quantitative purposes, and the information from multiple-stage mass spectrometry in an ion-trap mass analyzer contributed to fragmentation studies. Hydrophilic interaction liquid chromatography (HILIC) in a Fused-Core™ column using isocratic elution (acetonitrile:formic acid/ammonium formate buffer pH 4, 50 mM (60:40)) successfully analyzed this compound in less than 3 min. The HILIC system was coupled to heated electrospray-MS/MS using highly selective-selected reaction monitoring (H-SRM) to improve sensitivity and selectivity for the analysis of amprolium, after a simple sample treatment based on an “extract and shoot” strategy. Accurate mass measurements were performed to identify the interfering compound responsible for causing matrix ion enhancement in the signal of amprolium. The addition of l-carnitine (the interfering compound) (1 μg L⁻¹) to standards and sample extracts allowed the use of the external calibration method for quantitative purposes. The LC–MS/MS (H-SRM) method showed good precision (relative standard deviation, RSD, lower than 13%), accuracy and linearity and allowed the determination of amprolium down to the ppb level (LODs between 0.1 and 0.6 μg kg⁻¹).     

Residue analysis of glucocorticoids in bovine milk by liquid chromatography–tandem mass spectrometry

A sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 13 steroidal anti-inflammatory drugs in bovine milk is presented. Due to their weakly acid nature, analytes were separated by ion suppression reversed phase chromatography and detected in positive-ion mode by a high flow electrospray source. Dexamethasone-d4 was used as internal standard. The sample preparation was simple and reliable; it included acidic deproteinization of milk followed by sample enrichment and clean-up, utilizing a C18 solid phase extraction cartridge. Recoveries exceeded 70% with an intra-day precision not larger than 12%. The efficiency of the sample clean-up and internal standardization rendered negligible the matrix effect, estimated by comparing standard and matrix-matched calibration curves. A small-scale reconnaissance was carried out on several raw and whole fresh milk samples. A large number of analyzed samples showed a chromatographic peak, in the retention time window of cortisol, at levels included between its decision limit (CCα) and detection capability (CCβ). As a result of a heat-induced transformation, an isomeric product of triamcinolone was observed during the extract evaporation. Since this rearrangement might occur during the milk pasteurization process, LC-MS/MS and ¹H-NMR investigations were performed out to conclusively differentiate the two isomers. One- and two-dimensional proton NMR spectra were able to identify the transformation product as 9a-fluoro-11b,16a-trihydroxy-17b-hydroxymethyl-D-homoandrosta-1,4-diene-3,17a-dione.     

Determination of prostatic androgens in 10 mg of tissue using liquid chromatography–tandem mass spectrometry with charged derivatization

A practical liquid chromatography–tandem mass spectrometric (LC-MS-MS) method for the determination of prostatic 5-dihydrotestosterone (DHT) and testosterone (T) has been developed. The prostatic androgens were extracted with MeOH–H₂O (3: 7, v/v), purified with an Oasis HLB cartridge, derivatized with the permanently charged reagent 2-hydrazino-1-methylpyridine (HMP), and subjected to LC–MS-MS analysis using electrospray ionization (ESI) operated in the positive ion mode. The derivatization with HMP was very effective at increasing the detectability using the positive-ESI-MS. The method allowed the reproducible and accurate quantification of ng g^–1 tissue levels of prostatic androgens in 10 mg of tissue. That is, the intra- and inter-assay coefficients of variation were below 8.1 and 9.3%, respectively, and the analytical recoveries of the androgens were quantitative. The limits of quantitation for DHT and T were both 1.0 ng g^–1 tissue. The developed method was used to determine DHT and T in the prostates of patients with benign prostatic hyperplasia and prostate cancer, and satisfactory results were obtained.     

Determination of pharmaceuticals from different therapeutic classes in wastewaters by liquid chromatography–electrospray ionization–tandem mass spectrometry

A sensitive analytical method has been developed and validated for simultaneous determination of pharmaceuticals from different therapeutic classes, i.e. five sulfonamide (SA) and trimethoprim antimicrobials and the anti-inflammatory drug diclofenac, in effluent wastewaters at trace levels. Effluent samples from treatment of wastewater were enriched by solid-phase extraction (SPE) using the Waters Oasis HLB cartridge. The analytes were identified and quantified by reversed-phase liquid chromatography-tandem mass spectrometry operated in the selected reaction monitoring (SRM) mode, using positive electrospray ionization. The pharmaceuticals were, consequently, quantified both by use of isotopically labelled internal standards and by standard addition methods to address the issue of matrix effects related to signal suppression by co-eluting compounds. Average recoveries from fortified samples were usually >70%, with relative standard deviations below 20%. Method detection limits in wastewater matrices were between 7.0 and 10 ng L(-1). Identification points (IPs) were used for unequivocal identification of target analytes in real samples. Diclofenac, trimethoprim, and sulfamethoxazole were mainly detected, in the concentration range 10 to 400 ng L(-1), in effluent samples collected from four different sewage-treatment plants in Greece.     

Determination of selected pesticides in environmental water by employing liquid-phase microextraction and liquid chromatography–tandem mass spectrometry

An optimised extraction and cleanup method for the analysis of pesticide in natural water samples is presented. Sixteen pesticides of different polarity and from the different chemical classes (organophosphates, triazines, benzimidazoles, carbamates, carbamides, neonicotinoides, methylureas, phenylureas and benzohydrazides), most frequently used in Serbia, were selected for the analysis. Liquid-phase microextraction in a single hollow fibre (HF-LPME) has been applied for sample preparation. The concentrations of pesticides were determined using HPLC-MS/MS method with electrospray ionisation. The extraction behaviour and selection of the experimental conditions was predicted based on log D and pK _a values of targeted pesticides, which were calculated applying the computer software ACD/Labs PhysChem Suite v12. The influence of the donor pH and concentration of pesticides, organic phase composition as well as the extraction time on the extraction efficiency was investigated. Optimum extraction conditions were evaluated with respect to the investigated parameters of the extraction. The extraction method was validated for 10 out of 16 studied pesticides. Linear range of the pesticides was 0.1–5 μg L⁻¹ with the correlation coefficient from 0.991 to 0.9998, and the relative standard deviation for three standard measurements was between 0.2 and 11.8%. The limits of detections ranged from 0.026 to 0.237 μg L⁻¹ and the limits of quantifications from 0.094 to 0.793 μg L⁻¹. The optimised two-phase HF-LPME method was successfully applied for determination of moderately polar as well low-polar pesticides in the environmental water samples.     

Determination of pesticide residues in wine by membrane-assisted solvent extraction and high-performance liquid chromatography–tandem mass spectrometry

The determination of pesticides in food products is an essential issue to guarantee food safety and minimise health risks of consumers. A protocol based on membrane-assisted solvent extraction and liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) that allows the determination of 18 pesticides in red wine at minimum labour effort for sample preparation was developed and validated. Ten millilitres of wine were extracted using 100 μL of toluene filled in a non-porous polyethylene membrane bag which is immersed in the wine sample. After 150 min extraction under stirring, an aliquot of the extraction solution is analysed using HPLC-MS/MS. The limits of quantification ranged from 3 ng/L for Pirimicarb to 1.33 μg/L for Imidacloprid. Quantification by matrix-matched calibration provided relative standard deviations ≤16 % for most of the target pesticides. The linearity of calibration was given over three to four orders of magnitude, which enables the reliable measurement of a broad range of pesticide concentrations, and for each target pesticide, the sensitivity of the protocol meets the maximum residue levels set by legislations at least for wine grapes. Good agreement of results was found when the new method was compared with a standard liquid-liquid extraction protocol. In five wine samples analysed, Carbendazim and Metalaxyl were determined at micrograms per litre concentrations, even in some of the organic wines. Tebuconazol and Cyprodinitril were determined at lower abundance and concentration, followed by Spiroxamin and Diuron.     

Determination of multi-class pharmaceuticals in wastewater by liquid chromatography–tandem mass spectrometry (LC–MS–MS)

An analytical method for multi-class pharmaceuticals determination in wastewater has been developed and validated. Target compounds were: sulfonamides (sulfadiazine, sulfaguanidine, sulfamethazine, sulfamethoxazole), fluoroquinolones (ciprofloxacin, enrofloxacin, norfloxacin), diaminopyrimidine (trimethoprim), anaesthetic (procaine), anthelmintic (praziquantel and febantel), and macrolide (roxithromycin). The method involves pre-concentration and clean-up by solid-phase extraction (SPE) using Strata-X extraction cartridges at pH 4.0. Target analytes were identified and quantitatively determined by liquid chromatography–tandem mass spectrometry using multiple reaction monitoring (MRM). Recoveries were higher than 50% with relative standard deviation (RSD) below 18.3% for three concentrations. Only for sulfaguanidine was low recovery obtained. Matrix effect was evaluated using matrix-matched standards. The method detection limit (MDL) was between 0.5 and 5 ng L⁻¹ in spiked water samples. The precision of the method, calculated as relative standard deviation, ranged from 0.5 to 2.0% and from 1.4 to 8.3 for intra-day and inter-day analysis, respectively. The described analytical method was used for determination of pharmaceuticals in effluent wastewaters from the pharmaceutical industry.     

Determination of 19 drugs of abuse and metabolites in whole blood by high-performance liquid chromatography–tandem mass spectrometry

A high-performance liquid chromatography (LC)–tandem mass spectrometry (MS/MS) method has been developed and validated for the determination of 19 drugs of abuse and metabolites and used in whole blood. The following compounds were included: amphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylenedioxymethamphetamine, methamphetamine, cocaine, benzoylecgonine, morphine, 6-acetylmorphine, codeine, methadone, buprenorphine, norbuprenorphine, ketobemidone, tramadol, O-desmethyltramadol, zaleplone, zolpidem, and zopiclone. The sample pretreatment consisted of solid-phase extraction using mixed-mode columns (Isolute Confirm HCX). Deuterated analogues were used as internal standards for all analytes, except for ketobemidone and O-desmethyltramadol. The analytes were separated by a methanol/ammonium formate gradient using high-performance LC (Agilent HPLC 1100) with a 3 mm × 100 mm Varian Pursuit 3 C₁₈ column, 3-μm particle size, and were quantified by MS/MS (Waters Quattro micro tandem quadrupole mass spectrometer) using multiple reaction monitoring in positive mode. Two transitions were used for all analytes, except for tramadol and O-desmethyltramadol. The run time of the method was 35 min including the equilibration time. For all analytes, responses were linear over the range investigated, with R ² > 0.99. One-point calibration was found to be adequate by validation, thereby saving analysis of multiple calibrators. The limits of quantification (LOQs) for the analytes ranged from 0.0005 to 0.01 mg/kg. Absolute recoveries of the analytes were from 34 to 97%, except for zaleplone (6%). Both the interday precision and the intraday precision were less than 15% (20% at the LOQ) for all analytes, except buprenorphine, norburprenorphine, and zaleplone (less than 18%). Accuracy (bias) was within ±15% (±20% at the LOQ) for all analytes, except MDMA and O-desmethyltramadol (within ±19%). No ion suppression or enhancement was seen nor was suppression from coeluted analytes seen. Matrix effects were found to be less than 23% for all analytes, except zopiclone (64%). High-concentration and low-concentration quality control samples gave acceptable values, and the method has been tried in international proficiency test schemes with good results. The present LC-MS/MS method provides a simple, specific, and sensitive solution for the quantification of some of the most frequent drugs of abuse and their metabolites in whole blood. The quantification by LC-MS/MS was successfully applied to 412 forensic cases from October 2008 to mid February 2009, where 267 cases were related to zero-tolerance traffic legislation.     

Analysis of nucleosides and nucleotides in infant formula by liquid chromatography–tandem mass spectrometry

A method for the simultaneous analysis of nucleosides and nucleotides in infant formula using reversed-phase liquid chromatography–tandem mass spectrometry is described. This approach is advantageous for compliance testing of infant formula over other LC-MS methods in which only nucleotides or nucleosides are measured. Following sample dissolution, protein was removed by centrifugal ultrafiltration. Chromatographic analyses were performed using a C₁₈ stationary phase and gradient elution of an ammonium acetate/bicarbonate buffer, mass spectrometric detection and quantitation by a stable isotope-labelled internal standard technique. A single laboratory validation was performed, with spike recoveries of 80.1–112.9 % and repeatability relative standard deviations of 1.9–7.2 %. Accuracy as bias was demonstrated against reference values for NIST1849a certified reference material. The method has been validated for the analysis of bovine milk-based, soy-based, caprine milk-based and hydrolysed milk protein-based infant formulae.

Determination of multiple pharmaceutical classes in surface and ground waters by liquid chromatography–ion trap–tandem mass spectrometry

This paper describes development, optimization and application of analytical method for determination and reliable confirmation of nineteen pharmaceuticals from different therapeutic classes (antibiotics—β-lactams, cephalosporines, sulfonamides, macrolides and tetracyclines; benzodiazepines; antiepileptics and analgoantipyretics) in surface and ground waters at ng l⁻¹ levels. Water samples were prepared using solid-phase extraction and extracts were analyzed by liquid chromatography–ion trap–tandem mass spectrometry with electrospray ionization in both positive and negative ionization mode. The efficiency of ten different SPE cartridges to extract diverse compounds from water was tested. The pH-value of the water sample, the volume of elution solvent and the sample volume were optimized. Matrix effect, especially pronounced for cephalexin and metamizole, was eliminated using matrix-matched standards. It was determined that extraction should be performed at pH ∼ 7.5, i.e. without pH adjustment, and at pH 3, depending on the analyte. Azithromycin, doxycycline and acetylsalicylic acid must be extracted in acidic environment, whereas extraction of paracetamol, ampicillin, erythromycin and metamizole should be performed without pH adjustment. Repeatability of the method was generally lower than 20%. The estimated limits of detection were in the range from 0.15 to 12.46 ng l⁻¹. The method was applied to 26 water samples for monitoring of selected drug residues. Results revealed the presence of carbamazepine (80% of water samples), azithromycin (23%), as well as trimethoprim and paracetamol (both 15%). The most striking was the false positive signal of diclofenac in every analyzed water sample. Confirmation of the positive results was performed by repeated injection of the positive sample extracts using confirmatory method with additional transitions.