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.     

Advantages of LC–MS–MS compared to LC–MS for the determination of nitrofuran residues in honey

In the framework of developing analyses for exogenous contaminants in food matrices such as honey, we have compared data obtained by high-performance liquid chromatography coupled with mass spectrometry (LC–MS) to those provided by high-performance liquid chromatography and tandem mass spectrometry (LC–MS–MS). Initial results obtained with LC–MS showed that the technique lacked selectivity, which is why the method was validated by LC–MS–MS. This method involves a solid-phase extraction (SPE) of nitrofuran metabolites and nitrofuran parent drugs, a derivatization by 2-nitrobenzaldehyde for 17 h, and finally a clean-up by SPE. The data obtained show that the limits of detection varied between 0.2 and 0.6 μg kg⁻¹ for the metabolites and between 1 and 2 μg kg⁻¹ for nitrofuran parent drugs. The method was applied to different flower honeys. The results showed that nitrofurans (used as antibiotics) are consistently present in this matrix, the predominant compound being furazolidone. Figure Working bees     

LC–DAD and LC–ESI–MS Chromatographic Fingerprinting and Quantitative Analysis for Evaluation of the Quality of Huang-Lian-Jie-Du-Tang

LC–DAD coupled with electrospray tandem mass spectrometry (LC–ESI–MS–MS) has been used to evaluate the quality of the traditional Chinese medicine Huang-Lian-Jie-Du-Tang (HLJDT). Twenty-five chromatographic peaks were obtained from a C₁₈ analytical column by gradient elution with acetonitrile and formate buffer (containing 0.5% formic acid) at a flow rate of 1.0 mL min^?1. The linearity, precision, and accuracy of the data obtained were acceptable. Thirteen components were identified by ESI–MS, and seven of these were quantitatively analyzed by LC–DAD. The method was used to analyze ten batches of HLJDT, and both chromatographic fingerprints and quantitative data were used to evaluate the quality of the HLJDT. It was concluded that this LC–DAD–ESI–MS method enables more fully validated and complete evaluation and monitoring of the quality of HLJDT.     

Simultaneous and sensitive LC–MS/MS determination of tetrahydrocannabinol and metabolites in human plasma

Cannabis is not only a widely used illicit drug but also a substance which can be used in pharmacological therapy because of its analgesic, antiemetic, and antispasmodic properties. A very rapid and sensitive method for determination of ?⁹-tetrahydrocannabinol (THC), the principal active component of cannabis, and two of its phase I metabolites in plasma has been developed and validated. After solid-phase extraction of plasma (0.2 mL), the clean extracts were analyzed by tandem mass spectrometry after a 5-min liquid chromatographic separation. The linear calibration ranges were from 0.05 to 30 ng?mL^?1 for THC and 11-nor-?⁹-carboxy-tetrahydrocannabinol (THC-COOH) and from 0.2 to 30 ng?mL^?1 for ?⁹-(11-OH)-tetrahydrocannabinol (11-OH-THC). Imprecision and inaccuracy were always below 7 and 12 % (expressed as relative standard deviation and relative error), respectively. The method has been successfully applied to determination of the three analytes in plasma obtained from healthy volunteers after oral administration of 20 mg dronabinol.     

Quantitative Determination of Lobeline Hydrochloride in Rabbit Plasma by LC–MS–MS and Its Application

A sensitive and selective liquid chromatography tandem mass spectrometry method for quantitative determination of lobeline hydrochloride in rabbit plasma was developed and validated. After addition of triazolam as internal standard, protein precipitation by acetonitrile was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 column with acetonitrile-0.1% formic acid as mobile phase with gradient elution. Electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 338.1 → 315.8 for lobeline hydrochloride and m/z 342.9 → 308.0 for the IS. Calibration plots were linear over the range of 2–500 ng mL^?1 for lobeline hydrochloride in plasma. Lower limit of quantitation for lobeline hydrochloride was 2 ng mL^?1. Mean recovery of lobeline hydrochloride from plasma was in the range 97.5–102.3%. RSD of intra-day and inter-day precision were both <9%. This developed method is successfully used in pharmacokinetic study of lobeline hydrochloride in rabbit.     

A validated method for the determination of traces of UV filters in fish using LC–MS/MS

An analytical method for the determination of UV filter substances in fish tissue has been developed and validated using benzophenone-3, 3-(4-methylbenzylidene)-camphor, 2-ethylhexyl-2-cyano-3,3-diphenyl-2-propenoate and 2-ethylhexyl 3-(methoxyphenyl)-2-propenoate as target analytes. The fish fillets were homogenised and extracted by Soxhlet extraction. The extracts were run through a clean-up process including gel permeation chromatography followed by solid-phase extraction. Quantification of the compounds was performed using liquid chromatography with tandem mass spectrometric detection. Blank fish as well as spiked blank fish were analysed to validate the analytical method. The analytical method developed has the multiple advantages of enabling separation, simultaneous identification and quantification of each of the four selected compounds in a single run. Contamination of blank samples and abnormally high concentrations in spiked samples were avoided by taking extensive precautions during the fish preparation procedure. The method was validated in accordance with internationally accepted criteria, such as specificity, accuracy and repeatability. The combination of LC with tandem mass spectrometry ensures a high level of specificity. The accuracy of the method was reported as the mean recovery rate for the analytes in the sample matrix. Mean recoveries were in the range 86–108%. The precision is expressed as the relative standard deviation, and in all but one of the cases was 20% or below. The accuracy of the method allows residue analyses to be performed on biological matrices at ng/g levels. The determined limit of quantification for each analyte was 8 ng/g fish. For all spiking levels ≥8 ng/g, relative standard deviations were ≤ 20%.     

Simultaneous Quantitative Analysis of Shikonin and Deoxyshikonin in Rat Plasma by Rapid LC–ESI–MS–MS

The purpose of this article was to develop a rapid and robust LC–MS–MS method for quantifying shikonin and deoxyshikonin simultaneously in rat plasma using emodin as internal standard. The LC system consisted of an Agilent ZORBAX SB-C18 (1.8 μm, 250 × 4.6 mm, 20 °C) column. Elution with an isocratic mobile phase consisted of methanol/10 mM ammonium acetate in water/acetonitrile containing 0.05% formic acid (45:10:45, v/v/v) at a flow rate of 0.8 mL min^?1 yielded sharp, high-resolved peaks within 12 min. The lower limits of quantitation were 0.5 ng mL^?1 for shikonin, and 8 ng mL^?1 for deoxyshikonin. Correlation coefficient (r) values for the linear range of two analytes were greater than 0.99. Assay precision was <13% and accuracy was 87–99%. This newly developed method was used to the pharmacokinetic studies of the shikonin analogues in rats after intravenous administration (n = 4).     

Simultaneous determination of six major ergot alkaloids and their epimers in cereals and foodstuffs by LC–MS–MS

This paper describes a new and rapid method for accurate quantification of the six ergot alkaloids, ergometrine, ergotamine, ergosine, ergocristine, ergocryptine, and ergocornine, by liquid chromatography–tandem mass spectrometry (LC–MS–MS). The six ergot alkaloids studied have been defined by the European Food Safety Authority (EFSA) as among the most common and physiologically active ones. In addition, the method enables the quantification of the corresponding six epimers (ergo-inines) of these ergot alkaloids. This is of considerable importance in terms of the differences in toxicity of the isomeric forms. The method involves extraction under alkaline conditions using a mixture of acetonitrile and ammonium carbonate buffer followed by a rapid clean-up using dispersive solid-phase extraction with PSA (primary secondary amine) and a short chromatographic LC-run (21 min) with subsequent MS–MS detection. The method was developed and validated using ten different cereal and food samples. The major strength of the new method compared with previously published techniques is the simplicity of the clean-up procedure and the short analysis time. The limits of quantification were 0.17 to 2.78 μg kg⁻¹ depending on the analyte and matrix. Recovery values for the 12 ergot alkaloids spiked into ten different matrices at levels of 5, 50, and 100 μg kg⁻¹ were between 69 and 105% for 85 of 90 recovery measurements made over six days. Measurement uncertainty values were highly satisfactory. At a concentration level of 5 μg kg⁻¹ the expanded measurement uncertainty ranged from ±0.56 to ±1.49 μg kg⁻¹, at a concentration level of 100 μg kg⁻¹ the expanded measurement uncertainty ranged from ±8.9 to ±20 μg kg⁻¹. Both LOQs and measurement uncertainties were dependent on the analyte but almost independent of the matrix. The method performance was satisfactory when tested in a mini-intercomparison study between three laboratories from three different countries. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of 10 urinary BTEX metabolites using LC–MS/MS

Benzene, toluene, ethylbenzene, and xylene (BTEX) are a group of volatile organic compounds that are ubiquitous in the environment due to numerous anthropogenic sources. Exposure to BTEX poses a health hazard by increasing the risk for damage to multiple organs, neurocognitive impairment and birth defects. Urinary BTEX metabolites are useful biomarkers for the evaluation of BTEX exposure, because of the ease of sampling and their longer physiological half-lives compared with parent compounds. A method that utilizes LC–MS/MS was developed and validated for simultaneously monitoring of 10 urinary BTEX metabolites. During the sample preparation an aliquot of urine was diluted with an equal volume of 1% formic acid; internal standard solution was added, and then the sample was centrifuged and analyzed. The analytes were separated on the Kinetex-F5 column by applying a linear gradient, consisting of 0.1% formic acid and methanol. The method was validated according to the FDA Bioanalytical Method Validation Guidance for Industry. The mean method's accuracies of the spiked matrix were 81–122%; the inter-day precision ranged from 4 to 20%; the limits of quantitation were 0.5–2 μg/L. The method was used for the evaluation of baseline levels of urinary BTEX metabolites in 87 firefighters.     

Characterization of peptide–protein interactions using photoaffinity labeling and LC/MS

The combination of photoaffinity labeling (PAL) with modern mass spectrometric techniques is a powerful approach for the characterization of peptide–protein interactions. Depending on the analytical strategy applied, a PAL experiment can provide different levels of information ranging from the identification of interaction partners to the structural characterization of ligand-binding sites. On the basis of LC/MS data generated in the framework of the identification of the binding site of the neuropeptide corticotropin-releasing factor (CRF) on its binding protein (CRFBP), the key role of LC/MS in the characterization of photoadducts on different structural levels was demonstrated. Covalent photoadducts of rat CRFBP (rCRFBP) were obtained by PAL with different mono- and bifunctional benzophenone photoprobes designed on the basis of the sequence of the synthetic CRF fragment human/rat CRF^6–33 which binds to CRFBP with high affinity. In view of the stoichiometry, LC/MS analysis revealed that the photoadducts consisted of one molecule of photoprobe and one molecule of rCRFBP. For a further characterization of the photoadducts on the oligopeptide level, enzymatic digests of unlabeled rCRFBP and of the respective photoadduct were compared by peptide mapping monitored with LC/MS. Thereby, it was found that the photoprobe that contained the photophore at its N-terminus labeled the amino acid sequence rCRFBP(34–38), whereas the photoprobe that contained the photophore at its C-terminus labeled rCRFBP(12–26). On the basis of the characterization of the photoadduct formed by rCRFBP and the bifunctional photoprobe that contained photophores on both termini, semiquantitative comparison of different enzymatic digests was accomplished by application of the mass-selective multiple ion chromatogram strategy.     

LC–MS–MS Quantitative Determination of Ursolic Acid in Human Plasma and Its Application to Pharmacokinetic Studies

A sensitive and specific liquid chromatography–electrospray ionization-tandem mass spectrometry method has been developed and validated for the identification and quantification of ursolic acid in human plasma using glycyrrhetic acid as an internal standard. The method involves extraction with methyl tert-butyl ether. The analyte was separated on a C₁₈ column and analyzed in multiple reaction monitoring mode with a negative electrospray ionization interface using the [M–H]? ions, m/z 455.4 for ursolic acid and m/z 469.5 → m/z 425.5 for glycyrrhetic acid. The method was validated over the concentration range of 0.86–110.0 μg L^?1. The intra- and inter-day precisions were less than 13.53% relative standard deviation (RSD) and the accuracy was within ?4.76% in terms of relative error (RE). The lower limit of quantification was 0.86 μg L^?1 with acceptable precision and accuracy. There were almost no matrix effects. Recovery of ursolic acid from spiked drug-free plasma was higher than 68%. The method was used to study the pharmacokinetic profile of ursolic acid in human plasma after oral administration of Jieyu capsules.     

Stress Studies and Identification of Degradation Products of Cephalexin Using LC–PDA and LC–MS/MS

A stability indicating RP-HPLC method for cephalexin has been developed and validated to identify and characterize potential degradation products. Drug was subjected to hydrolytic (acidic, basic, and neutral), oxidative, thermal, and photolytic stresses as per ICH guidelines Q1A (R2) and Q1B. Chromatographic separation was achieved on C8 column with mixture of ammonium acetate buffer pH 4.5 and acetonitrile in gradient mode as a mobile phase with PDA detection. Specificity of the method was established by peak purity studies. Method was validated as per ICH guideline Q2 (R1) for accuracy, precision, linearity, sensitivity, and robustness. Kinetics for each degradation condition was studied with respect to order of reaction and rate constant. Method was found to comply with acceptance criteria of validation parameters with respect to specificity (peak purity greater than 0.999) linearity (r ² greater than 0.99), accuracy (% recovery in the range of 98–102%), and precision (% RSD not more than 2). A total of six degradation products were generated in different stress conditions; these were identified and structures were proposed using LC–MS/MS. Cephalexin undergoes degradation in almost all the conditions. The developed stability indicating method is suitable for analysis of stability samples as it adequately separates all degradation products. Degradation products generated in photolytic and oxidative conditions are reported for the first time.     

Isotopic labeling-assisted metabolomics using LC–MS

Metabolomics has emerged as the latest of the so-called “omics” disciplines and has great potential to provide deeper understanding of fundamental biochemical processes at the biological system level. Among recent technological developments, LC–HRMS enables determination of hundreds to thousands of metabolites over a wide range of concentrations and has developed into one of the most powerful techniques in non-targeted metabolomics. The analysis of mixtures of in-vivo-stable isotopic-labeled samples or reference substances with un-labeled samples leads to specific LC–MS data patterns which can be systematically exploited in practically all data-processing steps. This includes recognition of true metabolite-derived analytical features in highly complex LC–MS data and characterization of the global biochemical composition of biological samples. In addition, stable-isotopic labeling can be used for more accurate quantification (via internal standardization) and identification of compounds in different organisms.     

Simultaneous determination of eight adulterants in weight management supplements and herbs by HPLC–DAD and LC–MS/MS

An efficient HPLC–DAD method was developed for simultaneous determination of eight adulterants in weight management supplements and herbs. The eight adulterants were phenolphthalein, sibutramine, nuciferine, and five anthraquinone compounds including aloe-emodin, rhein, emodin, chrysophanol, and physcion. The analytes were ultrasonically extracted with 70% (v/v) methanol aqueous solution followed by centrifugation. The supernatant was subjected to HPLC analysis. A Phenomenex Luna C18 column was applied for chromatographic separation. The mobile phase was consisted of methanol and aqueous solution of 0.05% (v/v) phosphoric acid–0.025% (m/v) sodium dodecyl sulfonate. The flow rate of mobile phase was 0.8?ml?min^?1 with gradient elution. Clenbuterol and ibuprofen were used as internal standards. The retention times and the characteristic UV spectrograms were used for qualitative analysis. Quantifications were based on the internal standard curves. Good linearities (r?>?0.9996) for all analytes were obtained with the intra- and inter-day precision (n?=?6) ranging from 0.76 to 5.9% and 0.90 to 8.1%, respectively. The average recoveries from the spiked samples with different matrices varied from 73.4 to 114%. Validations were subsequently performed using LC–MS/MS. The proposed method successfully determined the target adulterants in eight commercial weight management supplements and five weight reducing herbs with satisfactory results.     

High-throughput protein precipitation method with 96-well plate for determination of doxepin and nordoxepin in human plasma using LC–MS/MS

The aim of this study was to establish a high-throughput and sensitive LC–MS/MS method for the determination of doxepin and its major active metabolite nordoxepin in human plasma. It has been designed for bioequivalence study for formulations containing 25 mg of doxepin. Doxepin and nordoxepin were extracted from human plasma samples by protein precipitation with acetonitrile by using protein precipitation 96-well plates. The analyte was separated using a Phenomenex Kinetex Biphenyl column (100 × 2.1 mm, 2.6 μm) using isocratic elution with a mobile phase of 20 mM ammonium formate (30%) and acetonitrile:methanol 3:7 v:v (70%) at a flow rate of 0.5 mL/min and an injection volume of 10 μL. The detection was performed using a triple quadrupole mass spectrometer by multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 280.4 → 107.0 and 283.4 → 235.0 for doxepin and doxepin-D3, respectively, and 266.3 → 106.9 and 269.3 → 235.0 for nordoxepin and nordoxepin-D3, respectively, in positive electrospray ionization mode. The total run time was 3.5 min. The method was validated over a concentration range of 50–10,000 pg/mL using a Triple Quad 4500 MS System (Sciex) for both analytes. The developed and validated method can be successfully used to study the bioequivalence/pharmacokinetics of doxepin and nordoxepin.     

Simultaneous determination of pinoxaden,cloquintocet-mexyl,clodinafop-propargyl ester and its major metabolite in barley products and soil using QuEChERS modified with multi-walled carbon nanotubes coupled with LC–MS/MS

Herein, a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method with multi-walled carbon nanotube (MWCNT) as a dispersive solid-phase extraction was developed for simultaneous determination of pinoxaden (PXD), cloquintocet-mexyl (CLM), clodinafop-propargyl ester (CPE) and its major metabolite (clodinafop, CP) in barley grass powder, barley grain, and soil using liquid chromatography–tandem mass spectrometry (LC–MS/MS). We found that MWCNT as an absorbent could improve the recoveries of the tested analytes, particularly CP, in complex matrices. Under the optimum conditions, the established MWCNT-modified QuEChERS coupled with LC–MS/MS method exhibited excellent linearity (R²) of ≥0.9912, low limits of detection (LODs) and quantification (LOQs) of 0.02–0.07 and 0.29–1.26 μg kg⁻¹, and acceptable recoveries of 80–130% with intra- and inter-day relative standard deviations (RSDs) < 10.5%. No strong matrix effect (ME) has been observed on the respective samples. The method was successfully applied to monitor the tested analytes in the representative field incurred samples. Conclusively, the proposed method is sensitive and reliable and could be used to monitor the residues of PDX, CLM, CPE, and CP in complicated agro-products and soil matrices.     

Validation of ELISA screening and LC–MS/MS confirmation methods for cocaine in hair after simple extraction

This article describes an easy and innovative extraction procedure for cocaine and its primary metabolite, benzoylecgonine (BE), from hair consisting of sonication with H₂O/0.1% formic acid for 4 h. The same extract was used for screening with an enzyme-linked immunoassay (ELISA) and confirmation by liquid chromatography–tandem mass spectrometry (LC–MS/MS). For the ELISA screening test a cutoff of 0.5 ng/mg was used according to the Society of Hair Testing recommendations. LC–MS/MS limits of detection (LODs) were established to be 10 pg/mg and 1 pg/mg for cocaine and BE, respectively. Linearity was obtained over a range of 0.2–5 ng/mg for BE (target analyte) in the ELISA screening test, while in the LC–MS/MS method the range was 0.10–10 ng/mg for cocaine and 0.01–10 ng/mg for BE. Intra- and interbatch coefficients of variation and mean relative errors were less than 20% for all analytes and concentrations studied. The validated ELISA and LC–MS/MS methods were applied to 48 hair samples and the results of both methods were compared; ELISA demonstrated a sensitivity and specificity of 89.2% and 10.8%.