LC–MS/MS in the routine clinical laboratory: has its time come?

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been increasingly used in routine clinical laboratories during the last two decades. The high specificity, sensitivity, and multi-analyte potential make it an ideal alternative to immunoassays or conventional high-performance liquid chromatography (HPLC). It also provides higher throughput than gas chromatography–mass spectrometry (GC–MS). LC–MS/MS also offers higher flexibility than immunoassays because LC–MS/MS assays are typically developed in-house. In addition, abundant information can be obtained from a single LC–MS/MS run which can produce a large amount of quantitative or qualitative data. In this review, typical LC–MS/MS clinical applications are presented, personal experiences are shared, and strengths and weakness are discussed. It is foreseeable that LC–MS/MS will become a key instrument in routine clinical laboratories.     

Liquid chromatography/negative ion atmospheric pressure photoionization mass spectrometry: a highly sensitive method for the analysis of organic explosives

Gas chromatography/mass spectrometry (GC/MS) is applied to the analysis of volatile and thermally stable compounds, while liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI‐MS) and liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) are preferred for the analysis of compounds with solution acid‐base chemistry. Because organic explosives are compounds with low polarity and some of them are thermally labile, they have not been very well analyzed by GC/MS, LC/APCI‐MS and LC/ESI‐MS. Herein, we demonstrate liquid chromatography/negative ion atmospheric pressure photoionization mass spectrometry (LC/NI‐APPI‐MS) as a novel and highly sensitive method for their analysis. Using LC/NI‐APPI‐MS, limits of quantification (LOQs) of nitroaromatics and nitramines down to the middle pg range have been achieved in full MS scan mode, which are approximately one order to two orders magnitude lower than those previously reported using GC/MS or LC/APCI‐MS. The calibration dynamic ranges achieved by LC/NI‐APPI‐MS are also wider than those using GC/MS and LC/APCI‐MS. The reproducibility of LC/NI‐APPI‐MS is also very reliable, with the intraday and interday variabilities by coefficient of variation (CV) of 0.2–3.4% and 0.6–1.9% for 2,4,6‐trinitrotoluene (2,4,6‐TNT). Copyright © 2008 John Wiley & Sons, Ltd.     

Comprehensive quantification of purine and pyrimidine metabolism in Alzheimer's disease postmortem cerebrospinal fluid by LC–MS/MS with metal-free column

The metabolome presence of nucleobases, nucleosides, nucleotides and related phosphorylated metabolites has been examined for Alzheimer's disease (AD). Although reversed-phase liquid chromatography tandem mass spectrometry (LC–MS/MS) has been used for the determination of these analytes, they were limited in chromatographic signal intensity and reproducibility owing to significant peak tailing caused by complexing with metallic cations and phosphate groups. In this work, we applied LC–MS/MS analysis with a metal-free column for comprehensive quantification of 40 analytes regarding to purine and pyrimidine metabolism in postmortem cerebrospinal fluid (pCSF) from AD patients. For the analytical column, an InertSustain AQ-C₁₈ metal-free PEEK column was used. MS detection was by electrospray positive ionization. The metal-free column allowed for sharp peak detection of highly polar metabolites within a running time of 17 min. In validation, the limits of detection (LOD), the limit of quantitation (LOQ) and recovery value using a pooled pCSF sample are 1–500 nM, 0.5–250 nM and a range of 53.1–144.0% (RSD ranged from 0.4 to 19.6%). The developed LC–MS/MS method utilizing a metal-free column provides an accurate quantification of some metabolites regarding purine and pyrimidine metabolism in pCSF samples obtained from AD patients.     

Study on the photostability of guaiazulene by high-performance liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry

The photostability of guaiazulene (1,4-dimethyl-7-isopropylazulene; GA), a natural azulenic compound used in cosmetic and health-care products, as well as in pharmaceutical preparations, was investigated in solution (methanol, ethanol, acetonitrile), by different techniques: gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography combined with atmospheric pressure chemical ionization mass spectrometry and UV detection (LC/APCI-MS and HPLC/UV). A solar simulator (xenon-arc lamp) was used as UV-A radiation source. The study involved: monitoring compound decomposition, identifying products of photodegradation (PPs), assessing the role of oxygen and evaluating the kinetics of the process. Minor PPs are volatile compounds and were characterized by GC/MS, while oligomeric polyoxygenated compounds, tentatively characterized on the basis of MS and MS/MS spectra, were found to be the main photoproducts. The photodegradation was found to be enhanced by the presence of oxygen; nevertheless, determination of the singlet oxygen quantum yield for GA gave a lower value than that for the reference standard Rose Bengal. The obtained results and the developed stability-indicating methods (GC/MS and LC/MS) are of interest for stability studies and/or quality control purposes of GA as raw material or cosmetic products.     

Quantification of rat brain neurotransmitters and metabolites using liquid chromatography/electrospray tandem mass spectrometry and comparison with liquid chromatography/electrochemical detection

Analytical methodology based on solid-phase extraction, polar reversed-phase liquid chromatography, and electrospray tandem mass spectrometry (LC/MS/MS) with isotope dilution was developed and validated for quantifying the neurotransmitters, dopamine and serotonin, and their major metabolites in brain tissue. Limits of detection (0.1-20 pg/mg tissue) were sufficient for analysis of multiple neurotransmitters in rat brain regions, including parietal cortex, hypothalamus, pituitary, substantia nigra, and striatum. Method performance was compared with contemporaneous measurements using a well-established procedure based on ion-pairing reversed-phase liquid chromatography and amperometric detection. The principal advantages of the LC/MS/MS method include a more robust sample purification procedure, an optimized chromatographic separation, and the qualitative and quantitative assurance that comes from coeluting isotopically labeled internal standards; however, sensitivity did not consistently improve upon that provided by amperometric detection. This methodology may be particularly useful for applications in which simultaneous determinations are required for drugs and their affected neurotransmitters in specific brain regions.     

Analysis of keto-enol tautomers of curcumin by liquid chromatography/mass spectrometry

Keto-enol tautomers of curcumin were confirmed by reversed-phase liquid chromatography(RPLC)/ hybrid quadrupole ion trap/time-of-flight mass spectrometry(QIT/TOFMS).Tautomers gave different MS/MS spectra in negative mode.Different mass spectra were also obtained by hydrogen/deuterium exchange LC/MS/MS in positive mode.Our results suggest that enol form is the major form in the solution(water/acetonitrile).     

Liquid Chromatography Tandem Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy of Magnesium (II) Gluconate Solution

Magnesium gluconate is a classical pharmaceutical compound used as a source of magnesium for the prevention and treatment of hypomagnesemia. To the best of our knowledge, a robust and reliable liquid chromatography tandem mass spectrometry technique has not yet been reported for the qualitative and quantitative analysis of magnesium gluconate. This study describes the method development for the LC–ESI–MS/MS analysis of magnesium gluconate using three different reversed-phase HPLC conditions (Method I–III) with comprehensive fragmentation pattern and the structural characterization by NMR spectroscopy. The LC–MS and NMR data were found in accordance with the structure of magnesium gluconate. When magnesium gluconate was dissolved in the acetonitrile and water–methanol solutions, it exists in situ in three different forms: magnesium gluconate itself, gluconic acid, and magnesium gluconate chelate with gluconic acid by a coordinate covalent bond. Method I exhibited pseudo-molecular ion peaks with more magnesium gluconate chelates with gluconic acid, while method II showed an adduct of magnesium gluconate with the solvent along with the molecular ion peak. There was no pseudo-molecular ion peaks found in method III. Thus, method III was found to be the more accurate, robust and reliable LC–MS method for the qualitative and quantitative analysis, structural characterization, and could also be suitable for the pharmacokinetic study of magnesium gluconate. The detailed fragmentation analysis might be useful for the structural characterization of unknown divalent organometallics.     

Peptide retention prediction applied to proteomic data analysis

A retention prediction model was developed for peptides separated in reversed-phase chromatography. The model was utilized to identify and exclude the false positive (FP) peptide identifications obtained via database search. The selected database included human proteins, as well as decoy sequences of random proteins. The FP peptide detection rate was defined either as number of retention time outliers, or random decoy sequence identifications. The FP rate for various MASCOT scores was calculated. The peptides identified in one-dimensional (1D) and two-dimensional (2D) liquid chromatography/mass spectrometry (LC/MS) experiments were validated by prediction models. Multi-dimensional LC was based on two orthogonal reversed-phase chromatography modes; prediction models were successfully applied for data filtering in both separation dimensions.     

Analysis of betamethasone, dexamethasone and related compounds by liquid chromatography/electrospray mass spectrometry

A reversed-phase high-performance liquid chromatography/electrospray ionisation mass spectrometry (HPLC/ESI-MS) method has been developed to conclusively differentiate the epimers betamethasone and dexamethasone and various esterification products (betamethasone and dexamethasone 21-acetate, betamethasone and dexamethasone 21-phosphate, betamethasone 17-valerate, betamethasone 21-valerate and betamethasone 17,21-dipropionate) in counterfeit drugs. Good separation with baseline resolution of all epimers or isomers was obtained on a Zorbax Eclipse XDB or Luna C8 column, using a step gradient with mobile phases of 0.05 M ammonium acetate and acetonitrile. Betamethasones can also be distinguished by the relative abundance of their m/z 279 ion in the positive electrospray tandem mass spectra. The LC/MS or LC/MS/MS method developed was successfully applied to the analysis of drug product samples, i.e. creams and tablets.     

Comparison of Three Analytical Methods for the Determination of Quinolones in Pig Muscle Samples

This work presents a comparison between three analytical methods developed for the simultaneous determination of eight quinolones regulated by the European Union (marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin, difloxacin, sarafloxacin, oxolinic acid and flumequine) in pig muscle, using liquid chromatography with fluorescence detection (LC–FD), liquid chromatography–mass spectrometry (LC–MS) and liquid chromatography-tandem mass spectrometry (LC–MS/MS). The procedures involve an extraction of the quinolones from the tissues, a step for clean–up and preconcentration of the analytes by solid-phase extraction and a subsequent liquid chromatographic analysis. The limits of detection of the methods ranged from 0.1 to 2.1 ng g⁻¹ using LC–FD, from 0.3 to 1.8 using LC–MS and from 0.2 to 0.3 using LC–MS/MS, while inter- and intra-day variability was under 15 % in all cases. Most of those data are notably lower than the maximum residue limits established by the European Union for quinolones in pig tissues. The methods have been applied for the determination of quinolones in six different commercial pig muscle samples purchased in different supermarkets located in the city of Granada (south-east Spain).

     

Comparison of LC–UV, LC–ELSD and LC–MS Methods for the Determination of Sesquiterpenoids in Various Species of Artemisia

Simple and specific analytical methods for the quantitative determination of sesquiterpenoids from various species of Artemisia plant samples were developed. By LC–UV, LC–ELSD, the separation was achieved by reversed-phase chromatography on a C18 column with water and acetonitrile both containing 0.025% trifluoroacetic acid as the mobile phase. In the LC–MS system, trifluoroacetic acid was replaced by 0.1% formic acid. The wavelength used for quantification of sesquiterpenoids with a diode array detector was 205 nm. The limits of detection by LC–MS was found to be 5, 10, 25, 50, 50 ng mL^?1. The limits of detection by LC–UV and LC–ELSD were found to be 5.0, 3.0, 100, 100, 7.5 μg mL^?1, by LC–UV and 50, 25, 30, 100 and 75 μg mL^?1 by LC–ELSD. LC–mass spectrometry coupled with electrospray ionization (ESI) interface is described for the identification and quantification of sesquiterpenoids in various plant samples. This method involved the use of the [M + H]⁺ ions of sesquiterpenoids in the positive ion mode with extractive ion monitoring.     

Characterization by mass spectrometry of an unknown polysiloxane sample used under uncontrolled medical conditions for cosmetic surgery

For a complete understanding of the raw material used for cosmetic surgery under uncontrolled medical conditions, an unknown sample of polydimethylsiloxanes has been investigated utilizing a combination of analytical techniques: pyrolysis/gas chromatography/mass spectrometry (Py/GC/MS), electrospray ionization (ESI)-MS, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)MS, and liquid chromatography (LC)/MS. Among these techniques, the LC/APCI-MS coupling allowed the fastest and more effective analysis. In addition, the complexity of the mass spectra deduced from these LC/MS experiments was simplified compared to the mass spectra obtained by MALDI-TOF. In this work, we have demonstrated how the LC/APCI-MS coupling applied to polydimethylsiloxane samples permits the full characterization of samples where end groups of different nature can be present in very small quantities.     

Comparison of Three Analytical Methods for the Determination of Quinolones in Pig Muscle Samples

This work presents a comparison between three analytical methods developed for the simultaneous determination of eight quinolones regulated by the European Union (marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin, difloxacin, sarafloxacin, oxolinic acid and flumequine) in pig muscle, using liquid chromatography with fluorescence detection (LC–FD), liquid chromatography–mass spectrometry (LC–MS) and liquid chromatography-tandem mass spectrometry (LC–MS/MS). The procedures involve an extraction of the quinolones from the tissues, a step for clean–up and preconcentration of the analytes by solid-phase extraction and a subsequent liquid chromatographic analysis. The limits of detection of the methods ranged from 0.1 to 2.1 ng g^?1 using LC–FD, from 0.3 to 1.8 using LC–MS and from 0.2 to 0.3 using LC–MS/MS, while inter- and intra-day variability was under 15 % in all cases. Most of those data are notably lower than the maximum residue limits established by the European Union for quinolones in pig tissues. The methods have been applied for the determination of quinolones in six different commercial pig muscle samples purchased in different supermarkets located in the city of Granada (south-east Spain).     

Analysis of antitumor active OSW-1 and its analogues by liquid chromatography coupled with electrospray and atmospheric pressure chemical ionization quadrupole mass spectrometry

Three cholestane glycosides including OSW-1 with antitumor activity and two new analogues with modified steroidal side chains, thienyl OSW-1 and silylated thienyl OSW-1, were synthesized. Analyses were performed using optimized, reversed-phase liquid chromatography (LC) with electrospray ionization and atmospheric pressure chemical ionization quadrupole mass spectrometry (MS). The ionization mode and polarity, cone voltage, and chromatographic conditions were evaluated. The optimum LC/MS conditions to obtain valuable ions, indispensable for identifying the structures, are described. The key fragmentation pathways, which will be useful for confirming the detailed structures of steroidal glycosides, are also proposed.     

A high-throughput monoamine oxidase inhibition assay using liquid chromatography with tandem mass spectrometry

A highly efficient method utilizing liquid chromatography with tandem mass spectrometry (LC/MS/MS) was developed and employed for high-throughput screening of compounds for monoamine oxidase (MAO) inhibition. The method used kynuramine as a common substrate for both MAO-A and MAO-B in incubations, and the 4-hydroxyquinoline (4-HQ) resulting from deamination of kynuramine followed by intramolecular condensation was analyzed using LC/MS/MS; formation of 4-HQ was used as the marker of MAO activity to evaluate the effects of test compounds. Isocratic liquid chromatography was applied to reduce the run time to 2 min. Because of the high specificity and sensitivity of detection of 4-HQ by LC/MS/MS, this method was able to use MAO enzymes at very low concentrations and to perform short incubations; as a result, consumable cost was minimized, and sample preparations were completely avoided. The inhibition data are highly reproducible, and the IC(50) values determined by the method are in good agreement with literature values. The results suggest that this method is very robust and can be used as a generic approach to screen for MAO inhibitors in drug discovery.     

Screening and determination of drugs in human saliva utilizing microextraction by packed sorbent and liquid chromatography–tandem mass spectrometry

This study presents a new method for collecting and handling saliva samples using an automated analytical microsyringe and microextraction by packed syringe (MEPS). The screening and determination of lidocaine in human saliva samples utilizing MEPS and liquid chromatography–tandem mass spectrometry (LC‐MS/MS) were carried out. An exact volume of saliva could be collected. The MEPS C₈‐cartridge could be used for 50 extractions before it was discarded. The extraction recovery was about 60%. The pharmacokinetic curve of lidocaine in saliva using MEPS‐LC‐MS/MS is reported. Copyright © 2013 John Wiley & Sons, Ltd.     

Forensic Identification of Dyes in Ballpoint Pen Inks Using LC–ESI–MS

The aim of this work was to develop a new technique using flow injection analysis combined with LC–ESI–MS which allows identification of dyes in ballpoint pen inks. A sample preparation procedure for the extraction of dyes from ballpoint pen strokes has been developed. The characteristic group of ions for each sample of 21 studied ballpoint pen inks corresponding to the present dyes has been determined using flow injection method. LC separation conditions for identified dyes have been optimized on reversed-phase sorbent based on silica gel. The best composition of the mobile phase for the dyes mixture LC separation was 0.1% aqueous formic acid and acetonitrile. Detection of dyes was carried out using mass spectrometry with electrospray ionization in positive and negative modes after reversed-phase liquid chromatography separation. Dye composition of ink was additionally confirmed comparing the data obtained from the literature. Flow injection analysis allows obtaining intensive ions of unknown strokes. It is difficult to get this information using only chromatographic methods, because dyes peak intensity can be low and noise of basic line high. Flow injection method allows distinguishing the analyzed 21 ballpoint pens by determining a characteristic set of dyes. The developed flow injection technique is very simple and quick. As a result, a novel approach for the identification of dyes in the ballpoint pen inks by flow injection analysis with LC–ESI–MS and UV detection without using standard dye samples has been established. It can be an effective alternative to the existing LC–DAD–MS and IR spectroscopy methods.     

Anisole, a new dopant for atmospheric pressure photoionization mass spectrometry of low proton affinity, low ionization energy compounds

Atmospheric pressure photoionization (APPI) is a novel method of ionization in liquid chromatography/mass spectrometry (LC/MS). It was originally developed in order to broaden the range of LC/MS ionizable compounds towards less polar compounds that cannot be analyzed by electrospray (ESI) and atmospheric pressure chemical ionization (APCI). Studies done thus far have shown that non-polar compounds that earlier were not ionizable in LC/MS can indeed be ionized by the use of APPI. However, the best ionization efficiency for low polarity samples has been achieved with low proton affinity (PA) solvents that are not suitable in reversed-phase LC (RP-LC). Here it is demonstrated that the signals for analytes with low proton affinities in acetonitrile can be increased 100-fold by using anisole as the dopant for APPI, which takes the sensitivity to the same level achieved in the analysis of high PA analytes.     

Synthesis of 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-N-methylhydrazino-2,1,3-benzoxadiazole (DAABD-MHz) as a derivatization reagent for aldehydes in liquid chromatography/electrospray ionization-tandem mass spectrometry

Benzofurazan derivatization reagent, 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-N-methylhydrazino-2,1,3-benzoxadiazole (DAABD-MHz), for aldehydes in liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS), was synthesized. DAABD-MHz reacted with aliphatic aldehydes under mild conditions. The generated derivatives were separated on a reversed-phase column and detected by ESI-MS/MS with detection limits of 30-60 fmol on-column. Upon collision-induced dissociation, a single and intense fragment ion at m/z 151 was observed. These results suggested that DAABD-MHz was suitable as a derivatization reagent in LC/ESI-MS/MS.     

Determination of the molecular weight of poly(ethylene glycol) in biological samples by reversed-phase LC–MS with in-source fragmentation

PEGylation has been widely used to improve the biopharmaceutical properties of therapeutic proteins and peptides. Previous studies have used multiple analytical techniques to determine the fate of both the therapeutic molecule and unconjugated poly(ethylene glycol) (PEG) after drug administration. A straightforward strategy utilizing liquid chromatography–mass spectrometry (LC–MS) to characterize high-molecular weight PEG in biologic matrices without a need for complex sample preparation is presented. The method is capable of determining whether high-MW PEG is cleaved in vivo to lower-molecular weight PEG species. Reversed-phase chromatographic separation is used to take advantage of the retention principles of polymeric materials whereby elution order correlates with PEG molecular weight. In-source collision-induced dissociation (CID) combined with selected reaction monitoring (SRM) or selected ion monitoring (SIM) mass spectrometry (MS) is then used to monitor characteristic PEG fragment ions in biological samples. MS provides high sensitivity and specificity for PEG and the observed retention times in reversed-phase LC enable estimation of molecular weight. This method was successfully used to characterize PEG molecular weight in mouse serum samples. No change in molecular weight was observed for 48 h after dosing.