Single Quadrupole MS Research Articles

Simultaneous Determination of Free Carnitine and Total Choline by Liquid Chromatography/Mass Spectrometry in Infant Formula and Health-Care Products: Single-Laboratory Validation

A single-laboratory validation study was conducted for a liquid chromatographic/mass spectrometric (LCIMS) method for the simultaneous determination of the free carnitine and total choline in milk-based infant formula and health-care products. The sample preparation used for both carnitine and choline was adapted from AOAC Official Method 999.14, with an acidic and enzymatic hydrolysis of esterified forms of choline. Carnitine and choline were quantified by ion-pair chromatography with single-quadrupole MS detection, using their respective deuterated internal standards. The repeatability relative standard deviation was < or =2.5 and 2.1%, respectively, for carnitine and choline. The intermediate reproducibility relative standard deviation was <4.7 and 2.4%, respectively, for carnitine and choline. The ranges of the average product-specific recoveries were 92-98 and 94-103%, respectively, for carnitine and choline. Choline concentration determined in infant formula reference material SRM 1846 was in agreement with the reference value. The proposed method was compared with the enzymatic methods for a range of products; good correlation (r = 0.99) was obtained, although a significant bias was observed for both analytes. The method, with a short chromatographic run time (7 min), is convenient for routine analysis to enhance analytical throughput and is a good alternative to enzymatic assays.

Cover Picture: Electrophoresis 10/2008

AbstractRegular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting‐edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two‐dimensional electrophoresis.“The present issue includes 29 manuscripts subdivided into three major parts: one part is devoted to instrumental and methodological advances, and two parts are providing an insight into up‐to‐date applications from the fields of natural products and food analysis on the one hand and biomedical and pharmaceutical analysis on the other hand. The approaches used comprise different modes of electroseparation methods such as CZE, packed column, monolithic column and open‐tubular CEC, MEKC, CIEF, CITP, different modes of ionization such as MALDI, ICP, and ESI, as well as a range of mass analyzers from simple single quadrupole MS to top of the range Q‐TOF instruments, providing MS‐MS and accurate mass features.”

Monitoring quinolone antibacterial residues in bovine tissues: extraction with hot water and liquid chromatography coupled to a single‐ or triple‐quadrupole mass spectrometer

A rapid and sensitive procedure for determining residues of seven quinolone antibacterials in bovine muscle, kidney and liver is presented. The method is based on the matrix solid-phase dispersion (MSPD) technique with hot water as extractant followed by liquid chromatography/single quadrupole mass spectrometry (LC/MS) or triple-quadrupole mass spectrometry (LC/MS/MS). After dispersing tissue samples on hydrazine sulfate treated sand, target compounds were eluted from the MSPD column by passing through it 4 mL of water heated at 100 degrees C. After pH adjustment and filtration, 200 and 5 microL of the aqueous extracts were respectively injected into the LC/MS and LC/MS/MS instruments. With the former instrument, MS data were acquired in the three-ion selected ion monitoring mode, while MS/MS data acquisition was performed in the multi-reaction monitoring mode by selecting two precursor ion to product ion transitions for each target compound. Hot water appeared to be an efficient extracting medium, since absolute recoveries of the analytes were 84-102%. Using norfloxacin (a quinolone not used in veterinary medicine) as surrogate internal standard, the accuracy of the method at three concentration levels equal to 0.5, 1 and 1.5 times the maximum residue limits (MRLs) set by the european union was 88-109% with relative standard deviations (RSDs) not higher than 7%. The use of LC/MS/MS allowed detection and quantification of the analytes in any tissue considered to be performed at concentrations by far lower than half of their MRLs. Vice versa, the single-quadrupole MS arrangement, while succeeding in monitoring quinolones in muscle tissue at the 0.5 MRL level, showed to be not sufficiently selective for unambiguous identification of some quinolones in kidney and liver.

Simultaneous determination of riboflavin, pyridoxine, nicotinamide, caffeine and taurine in energy drinks by planar chromatography-multiple detection with confirmation by electrospray ionization mass spectrometry

A new high-throughput method was developed to detect simultaneously riboflavin (Vitamin B 2), pyridoxine (Vitamin B 6), nicotinamide (Vitamin B 3), caffeine and taurine in energy drinks by multiple detection. Ten samples of energy drinks and six samples of beverages containing caffeine were prepared by degassing in an ultrasonic bath for 20 min. After chromatography, multi-wavelength scanning was performed by: (1) UV-absorbance measurement at 261 nm for nicotinamide and 275 nm for caffeine, (2) fluorescence measurement at 366/>400 and 313/>340 nm for riboflavin and pyridoxine, respectively, and (3) Vis-absorbance measurement at 525 nm for taurine, after post-chromatographic derivatization with ninhydrin reagent. Calibrations were linear or polynomial with determination coefficients r 2 > 0.999. Overall recoveries of the five compounds were between 81 and 106% at three different concentration levels. Repeatabilities (RSD, %) of all substances in matrix were determined to be between 0.8 and 1.5%. Intermediate precisions (RSD, %) ranged between 3.6 and 7.4% for riboflavin, 2.8 and 6.3% for nicotinamide, 2.5 and 4.4% for caffeine, 2.1 and 2.9% for taurine and 0.5 and 4.0% for pyridoxine at different concentration levels. Mass confirmation was performed by a single quadrupole MS in positive electrospray ionisation (ESI) scan mode for all substances except taurine (negative mode). This method offers a good alternative for routine analysis due to its simplicity and at the same time reliability.

Development of an analytical method for the determination of antidepressants in water samples by capillary electrophoresis with electrospray ionization mass spectrometric detection

A method for the quantitative determination of major antidepressants in aqueous matrices by CE using ESI-MS is presented. Several aqueous, nonaquoeus, and mixed aqueous/organic solvent BGEs including inorganic and organic acids were investigated with respect to their suitability for the separation of the selected analytes. Finally, due to the necessity to employ MS detection if the developed method should be suitable also for environmental samples, only MS-compatible electrolytes were taken into account. Based on this fact optimum results were obtained with a system consisting of 1.5 M formic acid and 50 mM ammonium formate in ACN/water (85/15). Linear calibration plots could be obtained for all solutes over a concentration range of almost two orders of magnitude, and the LODs achieved were in the range of 3-6 microg/L for trazodone and 39-43 microg/L for sertraline with the TOF instrument and the single quadrupole instrument in the SIM mode, respectively. This fact allowed the assumption that the presented method can be regarded as suitable for the determination of antidepressants even in the trace amounts commonly present in environmental samples. Spiking of river water and sewage plant effluent extracts with the selected solutes showed that no interferences from the matrix usually found in such samples can be expected. Finally the quantitative determination of the seven antidepressants in environmental samples was used to benchmark the performance of CZE coupled to a single quadrupole MS and a TOF-MS.

Application of Mass Spectrometry for Quantitative and Qualitative Analysis in Life Sciences

Due to its ability to analyze macromolecules, as well as polar and thermally labile low molecular weight compounds, mass spectrometry has become a very important tool in bioanalysis in recent years. The Life Sciences Mass Spectrometry Laboratory (LSMS) at the School of Pharmaceutical Sciences, University of Geneva, opened on April 1st, 2002. Its work is focused on the development and application of mass spectrometry for the qualitative and quantitative analysis of low molecular compounds and macromolecules present in biological fluids, tissues, organisms or cells. The instruments used for the research include: single quadrupole, triple quadrupole and hybrid MS instruments such as triple quadrupole linear ion traps; equipped with electrospray, nanoelectrospray, atmospheric pressure chemical ionization and matrix-assisted laser desorption.

Development of a Solid-Phase Extraction-HPLC/Single Quadrupole MS Method for Quantification of Perfluorochemicals in Whole Blood

A method for the determination of perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA) simultaneously with 10 closely related perfluorochemicals (PFCs) in human whole blood was developed and validated. PFOS and PFOA are used in various applications, for example, as surfactants and plastic additives, and are subject to environmental and health research due to their persistence. The main part of the data on PFCs in human blood is from serum samples, analyzed mainly by ion pair extraction followed by high-performance liquid chromatography (HPLC) and negative electrospray (ESI) tandem mass spectrometry (MS/MS). The analytical method developed here is suitable for human whole blood and involves solid-phase extraction (SPE) and HPLC negative electrospray single quadrupole mass spectrometry (HPLC/ES-MS). A whole blood aliquot was treated with formic acid and extracted on a octadecyl (C18) SPE column. The PFCs were isolated with methanol, and quantification was performed using single quadrupole mass spectrometry and perfluoroheptanoic acid as internal standard. Validation was performed in the range 0.3-194 ng/mL with recovery between 64 and 112% and limit of detection in the 0.1-0.5 ng/mL range for 11 of the 12 PFCs studied. We applied this method to 20 whole blood samples collected in 1997-2000 from the Swedish population in the ages 24-72. Eleven of the 12 PFCs were detected, and they were quantitatively and qualitatively confirmed using triple quadrupole LC/MS/MS analysis. PFOS, perfluorooctanesulfonamide, perfluorohexanesulfonate, PFOA and perfluorononanoic acid were quantified in all samples. In addition, perfluorohexanoic acid, perfluorodecanoic acid, perfluorodecanesulfonate, perfluoroundecanoic acid, perfluorododecanoic acid, and perfluorotetradecanoic acid were detected in some samples. This study shows that SPE and single quadrupole MS can be applied for extraction and quantification of PFCs in human whole blood, resulting in selectivity and low detection limits.

Identification and quantification of the hydroxyeicosatetraenoic acids, 20-HETE and 12-HETE, in the cerebrospinal fluid after subarachnoid hemorrhage

Purpose: The monohydroxylated metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE), is a potent vasoconstrictor of cerebral microvessels. 20-HETE formation is substantially elevated in the cerebral spinal fluid (CSF) in the rat subarachnoid hemorrhage (SAH) model. The presence of 20-HETE in human CSF has not been demonstrated. Therefore, it was the purpose of this study to determine if HETE metabolites are present in human CSF after SAH. Methods: CSF samples were collected daily from four SAH patients over 15 days. HETE metabolites were separated by HPLC with identification by ion-trap MS/MS and quantification via single quadrupole MS operating in negative single ion monitoring mode. Results: Two major metabolites were identified as 12-HETE and 20-HETE. 20-HETE maximal concentrations were 2.9 and 0.7 ng/ml at ∼70 h in the two patients with symptomatic cerebral vasospasm (SV) after SAH. Concentrations of 12-HETE in these patients peaked at 21.9 ng/ml and 2.8 ng/ml. Concentrations of 20-HETE and 12-HETE were non-detectible in the majority of the samples obtained from two matched SAH patients without SV. Conclusions: This study is the first to demonstrate that 20-HETE and 12-HETE are present in the CSF of SAH patients at physiologically relevant concentrations. Based on this information future prospective studies will allow for the delineation of the role of these metabolites in the pathogenesis of SAH.

Characterization of Sulfonamides by Flow Injection and Liquid Chromatography-Electrospray Ionization-Mass Spectrometry after Online Photoderivatization

The online photochemical identification of six sulfa compounds, sulfadiazine, sulfamerazine, sulfamethoxazole, sulfaisoxazole (SIX), sulfamoxole (SMX), and sulfamethizole, are investigated using flow injection and liquid chromatography (LC)-electrospray ionization-mass spectrometry (MS). Although the identification of some of the mentioned sulfonamides can be performed by recognizing their respected protonated molecules, more positive MS identification of many of these compounds is possible because they undergo various phototransformation processes to produce different product profiles. The LC separation and online photolysis of a mixture containing the geometric isomers SIX and SMX is such an example. With no photolysis, the MS spectra for SIX and SMX are virtually identical, showing primarily the sodiated molecule at m/z 290 with a relative abundance of 100% in addition to a few small peaks caused by fragments. With photolysis, SMX is found to form multiple major ions from 100 to 241 amu. However, SIX follows a similar fragmentation pathway either with or without photolysis. Online photochemistry should be a viable approach to extend the capabilities of LC instruments interfaced to a single quadrupole MS detector.

質量分析の法化学への応用

This paper reviews applications of mass spectrometry (MS) to forensic chemistry. Forensic chemistries have to cover the wide range of substances, and a court requests more sensitive and more reliable proof without limit. Thus MS is one of the most dependable technology for forensic chemistry. One of the most important field of forensic chemistry is analysis of drugs and toxicological substances. Especially, analysis of no-information samples so called general unknown screening (GUS) or systematic toxicological analysis (STA) is a challenging task for forensic chemists. The first topic is GUS of biological samples such as whole blood, plasma, urine, and stomach contents using MS. Gas-chromatography (GC)-MS occupied a position of “golden standard” of GUS for a long time. There is no doubt that LC-MS is currently competing with GC-MS to forensic chemistry. Though, developments of GUS procedure by LC-MS have just reported recently. Developments of GUS procedure by LC-MS are discussed about following points of view: interface types (electrospray ionization, ESI and atmospheric pressure chemical ionization, APCI); mass spectrometer (single quadrupole-MS, triplequadrupole-MS, tandem-MS, ion trap-MS, and time-of-flight-MS); mass spectral libraries; and chromatographic systems. Of course sample preparation or extraction is also essential for development of GUS, it lies outside of the scope of this review. Other forensic toxicological applications are discussed about following topics: GC-negative ion chemical ionization (NICI)-MS of benzodiazepines; LC-MS of gamma-hydroxybutyric acid (GHB); LC-inductively coupled plasma (ICP)-MS of phosphorus-containing amino acid type herbicides; LC-MS of quaternary ammonium herbicides; enanthioselective analysis of methamphetamine and related compounds by capillary-electrophoresis (CE)-MS; and single-dose detection of hypnotic sedatives. The other main field of forensic chemistry, analysis of industrial product, is also discussed as follows: ICP-MS of glass fragments, metal fragments of bullet, and dyes; LC-ICP-MS and ion chromatography-ICP-MS of arsenic compounds; pyrolysis-GC-MS of rubber and plastics; and explosive analysis by supercritical fluid chromatography-MS, GC-MS, and LC-MS. As mentioned above, application of MS to many scientific fields and development of analytical science helped forensic chemistry. Applications of MS for forensic chemistry, particularly GUS by LC-MS, might made contributions to other field such as environmental, pharmaceutical, and industrial chemistry.

Confirmatory analysis of sulfonamide antibacterials in bovine liver and kidney: extraction with hot water and liquid chromatography coupled to a single- or triple-quadrupole mass spectrometer.

A simple, specific, and rapid confirmatory method for determining 12 sulfonamide (SAs) antibacterials in bovine liver and kidney is presented. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography/mass spectrometry (LC/MS) with an electrospray ion source. The method was tailored for use with both single-quadrupole MS (I) and triple-quadrupole MS (II) instruments. After acidification and filtration of the aqueous extract, a 250-microL aliquot was injected into instrument I while only 25 microL was analyzed by instrument II. With instrument I MS data acquisition was performed in the selected ion monitoring (SIM) mode, selecting at least three ions for each target compound. With instrument II the selected reaction monitoring (SRM) mode with three fragmentation reactions for each compound was chosen. With the exception of sulfaquinoxaline (SQX), recovery of the analytes at the 50 ppb level in both liver and kidney was 72-96% with relative standard deviations (RSDs) ranging between 3 and 11%. The very poor recovery of SQX was due to its rapid enzymatic oxidation when in contact with the two tissues. With instrument I, limits of quantification (LOQs, S/N = 10) were 5-14 ppb of SAs. Even lower LOQs (1-8 ppb) were estimated by using instrument II, even though the extract volume analyzed was ten times lower than that with instrument I. With both matrices and using instrument I, severe ion signal suppression was experienced for the early-eluted SAs when trying to fractionate analytes by using a short chromatographic run time. This effect was traced to polar endogenous co-extractives eluted in the first part of the chromatographic run that interfered with gas-phase ion formation for SAs. Adopting more selective chromatographic conditions minimized this effect.

Selective determination of aromatic sulfonates in landfill leachates and groundwater using microbore liquid chromatography coupled with mass spectrometry.

Aromatic sulfonates (AS) are large-volume chemicals used in many technical processes of, for instance, the textile industry or construction. A LC/MS method for the selective determination of AS in environmental samples, based on a single-quadrupole MS, was developed and validated. The central point of this technique is the use of the compound-specific fragment ion SO3.- as marker for aromatic sulfonates. This negatively charged SO3 radical, together with the fact that AS undergo loss of SO2, allows screening for AS in complex matrixes, even in the presence of sulfate anions. Calibration curves generated from LC/MS data showed good linearity over 3 orders of magnitude, with an absolute limit of detection of approximately 1 ng. The relative standard deviation for mean areas obtained from reconstructed ion chromatograms ranged from 2.9 to 8.6%. Unlike UV detection, this LC/MS method gives similar response for both naphthalene- and benzene-sulfonates. The method presented was successfully applied to landfill leachates and groundwater, downstream of a landfill. Furthermore, this technique allowed identification of an unknown AS found in drain samples.

On-line nonaqueous capillary electrophoresis and electrospray mass spectrometry of tricyclic antidepressants and metabolic profiling of amitriptyline by Cunninghamella elegans.

An on-line nonaqueous capillary electrophoresis-electrospray mass spectrometry (ESI-MS) technique was developed using a commercial ion spray interface. The nonaqueous capillary electrophoresis ESI-MS system was used to profile tricyclic antidepressants of similar structures and mass-to-charge ratios. We found that pure methanol can be used as a sheath liquid to obtain stable ion spray from nonaqueous capillary electrophoresis. The flow rate of the coaxial nebulizing gas affected baseline signals, separation efficiency, and migration times. Other nonaqueous capillary electrophoresis operating conditions and electrospray parameters were optimized for enhanced baseline separation and high sensitivity detection. The effect of sample stacking on separation and detection was evaluated. The calculated detection limits were approximately 3 pg injected onto the capillary. ESI mass spectra of tricyclic antidepressants from a single quadrupole MS were obtained and elucidated. The information was used to propose fragmentation pathways of the tricyclic antidepressants. The method was also used to analyze the metabolites of amitriptyline produced by the fungus Cunninghamella elegans. Sixteen metabolites were detected and most of them were tentatively identified as demethylated and/or hydroxylated, and/or N-oxidized products.

Determination of acidic herbicides using liquid chromatography with pneumatically assisted electrospray ionization mass spectrometric and tandem mass spectrometric detection

Liquid chromatography–pneumatically assisted electrospray mass spectrometry with negative ionization has been used for the determination of acidic herbicides in ground water. Eighteen pesticides or pesticide degradation products belonging to several different groups of acidic herbicides (phenoxy acids, sulfonylureas, phenols, etc.) were covered in the study. Optimization of electrospray inlet conditions is described as well as results from investigations of the linearity of the detector response. Conditions for tandem mass spectrometry (MS–MS) detection of characteristic daughter ions formed by collision-induced dissociation (CID) of the parent ion are described and a comparison of obtainable instrument detection limits by single MS and MS–MS was made. Detection limits using MS in the selected ion monitoring (SIM) mode were generally in the order of 1 μg/l or below, whereas detection limits were three–four times higher using MS–MS detection. A principle of analysis is proposed based on single quadrupole MS as a method for quantitative determination followed by verification of positive findings by CID MS–MS. Application of the method for detecting acidic herbicides residues in a “real-world” ground water sample is demonstrated.

The characterisation of polyether ionophore veterinary drugs by HPLC-electrospray MS.

We undertake the determination of a wide range of veterinary drug residues in a range of animal products. Various screening analyses are employed, followed by HPLC-API (atmospheric pressure ionisation)-MS for the unequivocal confirmation of significant positives. EU legislation for the use of GC-MS as a confirmatory technique requires the successful monitoring of at least four diagnostic ions and although no such requirement exists for HPLC-MS confirmation, a similar requirement would seem appropriate. Until recently, reports describing the electrospray MS confirmation of residues of the polyether ionophores have been based on monitoring one or two ions. We have found that the addition of ammonium acetate to the HPLC mobile phase, in conjunction with 'cone voltage' or 'skimmer' assisted fragmentation, is a convenient way of producing additional diagnostic ions from polyether ionophore compounds, without compromising the overall sensitivity. Results for lasalocid, the most widely used compound, are presented. Electrospray MS data and acquisition parameters for lasalocid, monensin, narasin and salinomycin are described. The advantage of this analytical approach is that it may be used to generate confirmatory data using a single quadrupole MS system, without the need for advanced MS instrumentation, e.g., MS-MS.