Validation of a multi-analyte LC–MS/MS method for screening and quantification of 87 psychoactive drugs and their metabolites in hair

A multi-analyte method for the detection and quantification of 87 psychoactive drugs (antidepressants, antipsychotics, benzodiazepines, and z-drugs) in human hair has been developed and fully validated using the liquid chromatography–tandem mass spectrometry system. Due to the remarkable increase in requests of hair sample tests (such as for driver’s license renewals, child custody, DFA cases, and postmortem toxicology), we focused on the development of a rapid sample preparation. About 20 mg of hair samples, previously washed and cut into snippets, was ultrasonicated with 700 μl of methanol. Samples were then directly analyzed using a 4000 QTRAP (AB SCIEX, Foster City, CA, USA) with an electrospray ionization (ESI) Turbo V^TM Ion Source. The validation criteria parameters were satisfactory and in accordance with the international guidelines. All the compounds tested were successfully detected. One important aspect is the LODs in the low picogram per milligram concentration which may suggest a potential use of this method in cases of detection of single drug exposure. However, the LC–MS/MS method has been successfully applied for the analysis of postmortem cases (n?=?9).     

Pressurized-liquid extraction for determination of illicit drugs in hair by LC–MS–MS

An LC–MS–MS-based procedure for determination in hair of 14 different drugs of abuse belonging to the classes cocaine, amphetamine-like compounds, opiates, and hallucinogens has been developed. A pressurized-liquid extraction procedure was used and proved useful for quantitative recovery of all the analytes tested. This procedure, in conjunction with a simple decontamination step, performed to avoid false-positive samples, enabled the detection of all the analytes with LOQ ranging from 1.8 to 16 pg mg^?1 and accuracy varying from 85 to 111 %. The procedure was validated in accordance with the SOFT/AAFS guidelines and seems to be suitable for routine determination of the drugs tested in hair.     

Comparison of extraction efficiencies and LC–MS–MS matrix effects using LLE and SPE methods for 19 antipsychotics in human blood

Antipsychotic drugs are frequently associated with sudden death investigations. Detection of these drugs is necessary to establish their use and possible contribution to the death. LC–MS(MS) methods are common; however accurate and precise quantification is assured by using validated methods. This study compared extraction efficiency and matrix effects using common liquid–liquid and solid-phase extraction procedures in both ante-mortem and post-mortem specimen using LC–MS–MS. Extraction efficiencies and matrix effects were determined in five different blank blood specimens of each blood type. The samples were extracted using a number of different liquid–liquid extraction methods and compared with a standard mixed-mode solid-phase extraction method. Matrix effects were determined using a post-extraction addition approach—the blank blood specimens were extracted as described above and the extracts were reconstituted in mobile phase containing a known amount of analytes. The extraction comparison of ante-mortem and post-mortem blood showed considerable differences, in particular the extraction efficiency was quite different between ante-mortem and post-mortem blood. Quantitative methods used for determination of antipsychotic drugs in post-mortem blood should establish that there are no differences in extraction efficiency and matrix effects, particularly if using ante-mortem blood as calibrator.     

Quantitative determination of cationic modified polysaccharides on hair using LC–MS and LC–MS–MS

Cationic polysaccharides containing N-hydroxypropyl-N,N,N-trimethylammonium substituents are widely used as conditioning agents for hair-care products. A sensitive method has been developed for the quantitation of these polymers. After acidic extraction from hair the polysaccharides are hydrolyzed using trifluoroacetic acid. The cationic monoglycosides are determined using liquid chromatography–tandem mass spectrometry (LC–MS–MS). The developed method is independent of hair treatment. Even hair cut from test persons after customary hair wash can be analyzed. After treatment of natural and bleached hair tresses using a real-life treatment procedure 180 g and 300 g of polymer per gram hair were quantified, respectively. Additionally the fragmentation mechanism of the cationic N-hydroxypropyl-N,N,N-trimethylammonium group during electrospray ionization was investigated. A mass loss of 60 Da in combination with loss of a single charge is observed and associated with cleavage of trimethylamine and a proton. It is assumed that this process is promoted by the anionic counter-ion which might be hydroxide in an aqueous environment.     

Comparison of GC–MS and LC–MS methods for the analysis of antioxidant phenolic acids in herbs

Two methods were developed for the quantitative analysis of phenolic acids in herb extracts. The methods were based on liquid chromatography–time-of-flight mass spectrometry (LC–TOFMS) and gas chromatography–mass spectrometry (GC–MS). The methods were compared in terms of their linearity, repeatability, selectivity, sensitivity and the speed of the analysis. The sensitivity was good for both methods, with limits of detection of <80 ng/ml for most of the compounds. The relative standard deviations (RSD) of the peak areas were on average 7.2% for the LC–TOFMS method and 1.4% for the GC–MS method. Both methods were found to be suitable for the determination of the target analytes, although GC–MS was better suited to the quantitative determination of compounds present at low concentrations.     

Development and Validation of a LC–ESI–MS Assay for Determination of Icariin in Rat Plasma after Administration of Herba Epimedii

This paper reports a sensitive, specific, and stable chromatographic procedure with selective detection (electrospray mass spectrometry in selected-ion-monitoring mode) combined with simple and efficient sample preparation for determination of icariin in rat plasma after administration of Herba Epimedii. Separation of the analyte, possible endogenous compounds, and constituents of Herba Epimedii were accomplished on a 250 mm × 2.0 mm i.d. C₁₈ column by use of a rapid gradient. Ionization of icariin and clarithromycin (internal standard) was achieved by use of the electrospray interface in positive-ion mode. Conditions such as ionization mode, type of organic modifier, eluent additives, and Q-array potential were optimized to achieve good sensitivity and specificity of icariin detection. Response was a linear function of concentration over the range 0.2–20 ng mL⁻¹. The method is accurate and precise; within-batch and between-batch precision (CV) are <15%, and accuracy (RE) is better than ±15%. The method can be used for analysis of icariin in plasma after administration of Herba Epimedii or of traditional Chinese medicinal preparations containing Herba Epimedii.     

Determination of basic drugs of abuse in human serum by online extraction and LC–MS/MS

A new quantitation method for the determination of drugs of abuse (opiates, amphetamine and derivatives, cocaine, methadone and metabolites) in serum by using online extraction coupled to liquid chromatography (LC)–mass spectrometry (MS)/MS has been developed. The online extraction is carried out using two extraction columns simultaneously and one analytical column. One extraction column is loaded, while the other one is eluted by a gradient. The elution gradient also separates the analytes in the analytical column. For the sample preparation, serum is spiked with a mixture of deuterated analogues of the drugs. After protein precipitation with methanol/zinc sulphate, centrifugation, evaporation and reconstitution, the sample is injected into the LC system. The quantitation is based on the analysis of two multiple reaction monitoring transitions per drug. The recovery of the protein precipitation step is over 80% for all analytes. Intra- and interday precision, as relative standard deviation, is lower than 6%, and in the case of accuracy, RE is lower than 15%. Only the most polar analytes showed matrix effects. The limits of quantitation for the analysed compounds vary between 0.5 and 2.8 ng/mL. The developed method was used to quantify basic drugs in samples “from driving under the influence of drugs” cases. The results were compared with those obtained by using solid-phase extraction–GC–MS.     

Determination of multiclass pesticides in food commodities by pressurized liquid extraction using GC–MS/MS and LC–MS/MS

Pressurized liquid extraction (PLE) was applied to the simultaneous extraction of a wide range of pesticides from food commodities. Extractions were performed by mixing 4 g of sample with 4 g of Hydromatrix and (after optimization) a mixture of ethyl acetate:acetone (3:1, v/v) as extraction solvent, a temperature of 100°C, a pressure of 1000 psi and a static extraction time of 5 min. After extraction, the more polar compounds were analyzed by liquid chromatography (LC), and the apolar and semipolar pesticides by gas chromatography (GC); in both cases LC and GC were coupled with mass spectrometry in tandem (MS/MS) mode. The overall method (including the PLE step) was validated in GC and LC according to the criteria of the SANCO Document of the European Commission. The average extraction recoveries (at two concentration levels) for most of the analytes were in the range 70–80%, with precision values usually lower than 15%. Limits of quantification (LOQ) were low enough to determine the pesticide residues at concentrations below or equal to the maximum residue levels (MRL) specified by legislation. In order to assess its applicability to the analysis of real samples, aliquots of 15 vegetable samples were processed using a conventional extraction method with dichloromethane, and the results obtained were compared with the proposed PLE method; differences lower than 0.01 mg kg⁻¹ were found.     

Validation and use of three complementary analytical methods (LC–FLS, LC–MS/MS and ICP–MS) to evaluate the pharmacokinetics, biodistribution and stability of motexafin gadolinium in plasma and tissues

Liquid chromatography–fluorescence (LC–FLS), liquid chromatography–tandem mass spectrometry (LC–MS/MS) and inductively coupled plasma–mass spectrometry (ICP-MS) methods were developed and validated for the evaluation of motexafin gadolinium (MGd, Xcytrin) pharmacokinetics and biodistribution in plasma and tissues. The LC–FLS method exhibited the greatest sensitivity (0.0057 μg mL⁻¹), and was used for pharmacokinetic, biodistribution, and protein binding studies with small sample sizes or low MGd concentrations. The LC–MS/MS method, which exhibited a short run time and excellent selectivity, was used for routine clinical plasma sample analysis. The ICP–MS method, which measured total Gd, was used in conjunction with LC methods to assess MGd stability in plasma. All three methods were validated using human plasma. The LC–FLS method was also validated using plasma, liver and kidneys from mice and rats. All three methods were shown to be accurate, precise and robust for each matrix validated. For three mice, the mean (standard deviation) concentration of MGd in plasma/tissues taken 5 hr after dosing with 23 mg kg⁻¹ MGd was determined by LC–FLS as follows: plasma (0.025±0.002 μg mL⁻¹), liver (2.89±0.45 μg g⁻¹), and kidney (6.09±1.05 μg g⁻¹). Plasma samples from a subset of patients with brain metastases from extracranial tumors were analyzed using both LC–MS/MS and ICP–MS methods. For a representative patient, ≥90% of the total Gd in plasma was accounted for as MGd over the first hour post dosing. By 24 hr post dosing, 63% of total Gd was accounted for as MGd, indicating some metabolism of MGd.     

A simple and fast LC–MS/MS method for the simultaneous determination of tenofovir alafenamide and tenofovir in human plasma

A simple and fast liquid chromatography–tandem mass spectrometry method was established and validated for the simultaneous determination of tenofovir alafenamide (TAF) and tenofovir (TNF) in human plasma. A simple protein precipitation procedure was employed to extract analytes from plasma. Chromatographic separation was performed on an Eclipse Plus C₁₈ column utilizing a fast gradient elution starting with 2% of 2 mM ammonium acetate–formic acid (100/0.1, v/v) followed by increasing the percentage of acetonitrile. Detection was performed on a tandem mass spectrometer equipped with an electrospray ionization source operated in the positive ionization mode, using the transitions m/z 477.2 → m/z 346.1 for TAF and m/z 288.1 → m/z 176.1 for TNF. TAF-d₅ and TNF-d₇ were used as the internal standard of TAF and TNF, respectively. The method was validated in the concentration ranges 1.25–500 ng/mlfor TAF and 0.300–15.0 ng/ml for TNF with acceptable accuracy and precision.     

Validation of ELISA methods for search and quantification of β-n-methylamino-l-alanine in water and fish tissue

Water basins with low hydrodynamic activities can promote the growth and increase in algal biomass due to eutrophication, and toxic cyanobacteria species might then produce metabolites hazardous to human health. Over the last decade, a neurotoxic non-protein amino acid, (2S)-2-amino-3-(methylamino) propanoic acid, known as β-N-methylamino-L-alanine (BMAA), has become of particular interest because it has been hypothesised to be involved in progressive human neurodegenerative pathologies. This toxin can be found both in algal cells and free in water, as well as in some foods of aquatic and terrestrial origin. Analytical methods used for BMAA are often based on chromatography coupled with mass spectrometry, although these techniques involve long and expensive analysis. As the availability of a faster and cheaper screening method would be useful, we tested the only available Enzyme-Linked Immunosorbent Assay kit for BMAA evaluation and validated methods to verify their reliability for the analysis of water and fish muscle. For both matrices, we determined adequate selectivity and repeatability (relative standard deviation < 6%), with recoveries from 70% to 83% at the tested spiking levels; the methods were also robust. These data appear in contrast to a previous evaluation carried out on the same kit in 2013, although this might depend on an improvement to the kit performance. We can conclude that a preliminary determination of BMAA in water, and also in fish tissue after an adequate extraction procedure, can be performed efficiently with the tested kit, which provides for easier monitoring of this dangerous toxin.     

LC–MS methods combination for identification and quantification of trans-sinapoylquinic acid regioisomers in green coffee

The present study aims to both identify and quantify trans-sinapoylquinic acid (SiQA) regioisomers in green coffee by combined UHPLC-ESI-QqTOF-MS/MS and UHPLC-ESI-QqQ-MS/MS methods. Among the various mono-acyl chlorogenic acids found in green coffee, SiQA regioisomers are the least studied despite having been indicated as unique phytochemical markers of Coffea canephora (known as Robusta). The lack of commercially available authentic standards has been bypassed by resorting to the advantages offered by high-resolution LC–MS as far as the identification is concerned. SiQA regioisomers have been identified in several samples of Robusta and Coffea arabica (known as Arabica) commercial lots from different geographical origin and, for the first time, in different samples of coffee wild species (Coffea liberica and Coffea pseudozanguebariae). Quantification (total SiQA ranging from 3 to 5 mg/100 g) let to reconsider these chlorogenic acids as unique phytochemical markers of Robusta being present in the same quantity and distribution in C. liberica as well. Gardeniae Fructus samples (fruits of Gardenia jasminoides) have additionally been characterized as this matrix is recognized as one of the few naturally occurring SiQA sources. The SiQA regioisomer content (total SiQA about 80 mg/100 mg) fully supports the proposal to use this matrix as a surrogate standard for further studies.     

The identity confirmation of 99gTc-DMSA complexes by using NMR and HPLC–MS/MS methods

The analyses of ^99gTc-DMSA complexes prepared under alkali and acidic reactions were reported. Modern analytical, separation and spectral methods such as NMR (¹H-NMR, ¹³C-NMR, APT, COSY and HSQC) and Q-TOF HPLC–MS/MS system with ESI were employed to determine the identity and characterization of the products. The structure of ^99gTc(V)DMSA was clearly confirmed and its fragmentation path in negative and positive ionisation mode was suggested. The effect of ascorbic acid and new alternative labelling with the use of NH ₄ ^99g TcOCl₄ was examined. Surprisingly, ^99gTc(III)DMSA complex was not formed under acidic reaction conditions. ^99gTc(V)DMSA complex was the main reaction product under both experimental conditions. This result suggests the key role of ^99g/99mTc concentration during the process of radiopharmaceuticals preparation.     

Application of a simple and sensitive GC–MS method for determination of morphine in the hair of opium abusers

Thirty hair samples were collected from male opioid abusers for whom the presence of morphine in their urine samples was confirmed by thin layer chromatography (TLC). The hair samples were decontaminated by washing with isopropanol, deionized water, and isopropanol, dried at room temperature, and cut into small pieces. Samples of the latter (30 mg ) were digested by incubation in a mixture of methanol–trifluoroacetic acid (9:1) for 18 h at 45 °C and sonicated to improve the extraction process. The methanolic phase was evaporated to dryness under a stream of nitrogen at 50 °C. The sample was derivatized by addition of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) and 1% trimethyliodosilane (TMIS) at 70 °C for 20 min, with sonication. Derivatized samples (1 L) were injected into a gas chromatograph–mass spectrometer (GC–MS) system fitted with a capillary column; the Finnigan MS was operated in SIM mode. Naltrexone was used as internal standard (IS). The masses of the ions selected for morphine and naltrexone were 429 and 557, respectively. The limit of quantitation was set at 0.03 ng mg^–1 hair. By using the above procedure we detected morphine in all the samples examined, in the concentration range 0.26–10.31 ng mg^–1 hair.     

Rapid and simple method for determination of hexabromocyclododecanes and other LC–MS–MS-amenable brominated flame retardants in fish

In this study, a novel analytical approach for simultaneous determination of hexabromocyclododecane isomers (HBCDs), tetrabromobisphenol A (TBBPA), three brominated phenols, and four hydroxylated derivatives of polybrominated diphenyl ethers (OH-PBDEs) was developed and validated for muscle tissue of both lean and fatty fish. The rapid, simple, and high-throughput sample-preparation procedure was based on acetonitrile extraction then purification by dispersive solid-phase extraction (d-SPE) with a combination of C₁₈ and primary–secondary amine (PSA) sorbents. Ultra-high performance liquid chromatography (UHPLC) coupled to tandem mass spectrometry (MS–MS) was used for identification and quantification of the analytes. Method recovery for both matrices ranged from 80 to 115 % with relative standard deviations (RSDs) <13 % for all analytes. Limits of quantification (LOQs) were in the range 0.1–1 μg kg^?1 wet weight. The validated method was used for analysis of brominated compounds in 32 fish and five bivalve samples collected from different European markets within the monitoring survey organized in the framework of the CONffIDENCE project. Of the 12 targeted analytes, only α-HBCD, 2,4-dibromophenol (2,4-DBP), and 2,4,6-tribromophenol (2,4,6-TBP) were quantified in the samples. α-HBCD was found in six fish samples (herring and mackerel) in the range of 0.8–2.5 μg kg^?1 wet weight. 2,4-DBP and 2,4,6-TBP were found in three blue mussel samples in the range of 19.6–43.5 and 2.3–7.5 μg kg^?1 wet weight, respectively.     

Combined solid-phase extraction and 2D LC–MS for characterization of the neuropeptides in rat-brain tissue

A comprehensive two-dimensional capillary liquid chromatographic (2D LC) method has been established for determination of neuropeptides in rat brain tissue. Rats were exposed to different levels of stress before sacrificing and the aim of this study was to design a powerful separation and detection technique capable of characterizing differences between cerebral neuropeptide expression as a function of stress level. Rat brain samples were homogenized and subjected to clean-up by solid-phase extraction (SPE) on both a reversed-phase (C₁₈) and a weak cation-exchange (CBA) cartridge. The samples were divided in two fractions (A and B) depending on retention on the CBA column. Subsequently, 50 L of the sample were injected on to a strong cation exchanger (SCX) at a mobile phase pH of 3, which enabled preconcentration of positively charged compounds. The trapped compounds were eluted using step gradients of ammonium formate in water–ACN (90:10, v/v). Before enrichment in the second dimension, the eluate from the first dimension was diluted with water containing 0.1% TFA. The compounds eluting from the first dimension were trapped in the second dimension using a dual precolumn system consisting of two short capillary columns packed with Kromasil C₁₈, 10 m particles. Subsequently, the trapped compounds were backflushed on to a 10 cm long, 320 m I.D. analytical column packed with Kromasil C₁₈ 3.5 m particles, on which they were efficiently separated. Detection was performed using an ion-trap mass spectrometer (ITMS) in both the MS and the MS–MS mode. Comparison of base-peak chromatograms (BPC) from MS analysis of stressed and non-stressed rats clearly revealed several differences in neuropeptide expression. The MS–MS data obtained combined with Mascot software were employed for peptide identification.