Operational variables in high-performance liquid chromatography-electrospray ionization mass spectrometry of peptides and proteins using poly(styrene-divinylbenzene) monoliths

Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) utilizing monolithic poly(styrene-divinylbenzene) columns was optimized for the coupling to electrospray ionization mass spectrometry (ESI-MS) by the application of various temperatures and mobile phase additives during peptide and protein analysis. Peak widths at half height improved significantly upon increasing the temperature and ranged from 2.0 to 5.4 s for peptide and protein separations at 70 degrees. Selectivity of peptide elution was significantly modulated by temperature, whereas the effect on proteins was only minor. A comparison of 0.10% formic acid (FA), 0.050% trifluoroacetic acid (TFA), and 0.050% heptafluorobutyric acid (HFBA) as mobile phase additives revealed that highest chromatographic efficiency but poorest mass spectrometric detectabilities were achieved with HFBA. Clusters of HFBA, water, and acetonitrile were observed in the mass spectra at m/z values >500. Although the signal-to-noise ratios for the individual peptides diverged considerably both in the selected ion chromatograms and extracted mass spectra, the average mass spectrometric detectabilities varied only by a factor of less than 1.7 measured with the different additives. Limits of detection for peptides with 500 nl sample volumes injected onto a 60 mm x 0.20 mm monolithic column were in the 0.2-13 fmol range. In the analysis of hydrophobic membrane proteins, HFBA enabled highest separation selectivity at the cost of lower mass spectral quality. The use of 0.050% TFA as mobile phase additive turned out to be the best compromise between chromatographic and mass spectrometric performance in the analysis of peptides and proteins by RP-HPLC-ESI-MS using monolithic separation columns.     

Improved detection of multi-phosphorylated peptides in the presence of phosphoric acid in liquid chromatography/mass spectrometry

In contrast to lower phosphorylation states (e.g. the tryptic monophosphopeptide FQpSEEQQQTEDELQDK from bovine beta-casein), the specific detection of multi-phosphorylated peptides (e.g. the tetraphosphopeptide RELEELNVPGEIVEpSLpSpSpSEESITR from tryptic digestion of bovine beta-casein) has often been problematic for liquid chromatographic mass spectrometric (LC/MS) analysis owing to their high affinity for adsorption to exposed surfaces. We observed an enhancement in the overall detection of phosphopeptides on addition of phosphoric acid (0.1-1.0%) to the sample solution; a 10-fold increase in sensitivity was determined for the detection of two tryptic phosphopeptides and also a significant improvement in the detection of the tetraphosphopeptide. Using capillary LC with ion trap tandem MS for detection and identification, the achievable detection limits were 50 fmol and 50 pmol for the monophosphopeptide and the tetraphosphopeptide, respectively. Phosphoric acid is believed to act as a blocking agent to available silanol groups on both the silica capillary surface and the C(18)-bonded stationary phase silica surface.     

Short capillary ion-pair high-performance liquid chromatography coupled to electrospray (tandem) mass spectrometry for the simultaneous analysis of nucleoside mono-, di- and triphosphates

A liquid chromatography/mass spectrometry (LC/MS) method for the analysis of complex mixtures of nucleoside mono-, di- and triphosphates has been developed. A short capillary column (35mm x 0.3mm i.d.) was operated under ion-pair high-performance liquid chromatography conditions and hyphenated to (negative) electrospray (tandem) mass spectrometry. As such, the separation of 12 nucleotides was performed by a binary gradient elution using CH(3)OH/H(2)O and N,N-dimethylhexylamine (N,N-DMHA) as ion-pairing agent. The influence of different N,N-DMHA concentrations on the chromatographic and mass spectrometric performance was evaluated to achieve optimal LC/MS conditions. In addition it was demonstrated that a controlled admission of ammonium dihydrogen phosphate (NH(4)H(2)PO(4)) improved both chromatographic performance and mass spectrometric detection. Because the system was hyphenated to an orthogonal designed electrospray interface (Z-spraytrade mark), long acquisition times were possible without loss of sensitivity.     

Combination of liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry for the detection of 21 anabolic steroid residues in bovine urine

For the detection of anabolic steroid residues in bovine urine, a highly sensitive liquid chromatographic/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method was developed using both positive and negative ionization. For four compounds the ESI mode was not sensitive enough and gas chromatographic/mass spectrometric GC/MS detection was therefore still necessary as a complementary method. The sample clean-up consisted of solid-phase extraction (SPE) on a C(18) column followed by enzymatic hydrolysis and a second solid-phase extraction on a combination of a C(18) and a NH(2) column. After this last SPE clean-up, the eluate was split into two equal aliquots. One aliquot was further purified and after derivatization used for GC/MS analysis. The other aliquot was analyzed with LC/MS/MS in both ESI+ and ESI- modes. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) were between 0.16 and 1 ng ml(-1) for the compounds detected with the LC/MS/MS method. The developed method is used in routine analysis in our laboratory.     

Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is often used to enhance the detection sensitivity. Derivatization improves the chromatographic separation, and enhances the mass spectrometric ionization efficiency and MS/MS detectability. In this review, an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS is presented, focusing on the applications to low molecular weight compounds.     

Peptide mapping with liquid chromatography using a basic mobile phase

A new peptide mapping with liquid chromatography (LC) using an ammonia-containing basic mobile phase was reported. As compared with a method under a traditional acidic condition with a mobile phase containing trifluoroacetic acid (TFA) or formic acid (FA), the new method exhibited excellent overall performance: it was advantageous over the TFA method in terms of the ultraviolet (UV) and mass spectrometry (MS) sensitivities and the sequence coverage for a tryptic map; it was superior to the FA method in terms of the UV sensitivity, the sequence coverage and the separation capacity. Due to a significant difference in the chromatographic selectivity, several important peptide mapping applications that were sometimes difficult to be conducted previously could now be carried out using the new method. For example, the baseline separation of peptides from the corresponding deamidated products could be achieved with confidence using the new method, a critical pre-requisite for definitive identification and quantification of the deamidation products with LC/MS. No on-column deamidation was observed with the conditions used for the separation. Complementary and confirmative information about a protein could be obtained by running its proteolytic digest under both the basic and acidic conditions.     

Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes

Two new ultra-high performance liquid chromatography (UHPLC) methods for analyzing 21 selected antivirals and their metabolites were optimized, including sample preparation step, LC separation conditions, and tandem mass spectrometry detection. Micro-solid phase extraction in pipette tips was used to extract antivirals from the biological material of Hanks balanced salt medium of pH 7.4 and 6.5. These media were used in experiments to evaluate the membrane transport of antiviral drugs. Challenging diversity of physicochemical properties was overcome using combined sorbent composed of C₁₈ and ion exchange moiety, which finally allowed to cover the whole range of tested antivirals. For separation, reversed-phase (RP) chromatography and hydrophilic interaction liquid chromatography (HILIC), were optimized using extensive screening of stationary and mobile phase combinations. Optimized RP-UHPLC separation was carried out using BEH Shield RP18 stationary phase and gradient elution with 25 mmol/L formic acid in acetonitrile and in water. HILIC separation was accomplished with a Cortecs HILIC column and gradient elution with 25 mmol/L ammonium formate pH 3 and acetonitrile. Tandem mass spectrometry (MS/MS) conditions were optimized in both chromatographic modes, but obtained results revealed only a little difference in parameters of capillary voltage and cone voltage. While RP-UHPLC-MS/MS exhibited superior separation selectivity, HILIC-UHPLC-MS/MS has shown substantially higher sensitivity of two orders of magnitude for many compounds. Method validation results indicated that HILIC mode was more suitable for multianalyte methods. Despite better separation selectivity achieved in RP-UHPLC-MS/MS, the matrix effects were noticed while using both chromatographic modes leading to signal enhancement in RP and signal suppression in HILIC.     

Quantitative determination of folic acid in multivitamin/multielement tablets using liquid chromatography/tandem mass spectrometry

Two different isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods for the quantitative determination of folic acid (FA) in multivitamin/multielement tablets are reported. These methods represent distinct improvements in terms of speed and specificity over most existing microbiological and chromatographic methods for the determination of FA in dietary supplements. The first method utilizes an aqueous/organic-based extraction solvent combined with positive-ion mode LC/MS/MS detection of protonated [M + H]+ FA molecules and the second method utilizes a pure aqueous-based extraction solvent combined with negative-ion mode LC/MS/MS detection of deprotonated [M - H]- FA molecules. The LC/MS/MS methods exhibit comparable linear dynamic ranges (> or =3 orders of magnitude), limits of detection (0.02 ng on-column) and limits of quantification (0.06 ng on-column) for FA. Two methods employing different extraction and different MS detection modes were developed to allow method cross-validation. Successful validation of each measurement procedure supports the use of either method for the certification of FA levels in dietary supplements. The accuracy and precision of each measurement procedure were evaluated by applying each method to the quantitative determination of FA in a NIST standard reference material (NIST SRM 3280 multivitamin/multielement tablets). The FA measurement accuracy for both methods was > or =95% (based on the manufacturer's assessment of the FA level in SRM 3280) with corresponding measurement precision values (% RSD) of approximately 1%.     

Multi-residue liquid chromatography/tandem mass spectrometry method for the detection of non-steroidal anti-inflammatory drugs in bovine muscle: optimisation of ion trap parameters

A multi-residue liquid chromatography/tandem mass spectrometry method (LC/MS2) was developed for the detection of the non-steroidal anti-inflammatory drugs acetylsalicylic acid (via the marker residue salicylic acid), flunixin, phenylbutazone, tolfenamic acid, meloxicam and ketoprofen, in bovine muscle. After extraction of the bovine muscle with acetonitrile, the cleanup was performed using a Oasis HLB column. The evaporated eluate was reconstituted and analysed by LC/MS2. To obtain optimal detection of salicylic acid and phenylbutazone, the ion trap mass spectrometric parameters activation q and maximum ion injection time, respectively, were optimised. The activation q for salicylic acid was increased to obtain reliable detection of both salicylic acid and its product ion. The maximum ion injection time for the time segment containing phenylbutazone was decreased since there were not enough scans across the chromatographic peak of this compound. The multi-residue method was able to detect the different analytes below or at the maximum residue limit (MRL) or minimum required performance limit (MRPL) or, in the case of phenylbutazone and ketoprofen, at 100 and 20 microg kg(-1), respectively.     

Feasibility of a liquid-phase microextraction sample clean-up and liquid chromatographic/mass spectrometric screening method for selected anabolic steroid glucuronides in biological samples

Anabolic androgenic steroids (AAS) are metabolized extensively in the human body, resulting mainly in the formation of glucuronide conjugates. Current detection methods for AAS are based on gas chromatographic/mass spectrometric (GC/MS) analysis of the hydrolyzed steroid aglycones. These analyses require laborious sample preparation steps and are therefore time consuming. Our interest was to develop a rapid and straightforward method for intact steroid glucuronides in biological samples, using liquid-phase microextraction (LPME) sample clean-up and concentration method combined with liquid chromatographic/tandem mass spectrometric (LC/MS/MS) analysis. The applicability of LPME was optimized for 13 steroid glucuronides, and compared with conventional liquid-liquid extraction (LLE) and solid-phase extraction (SPE) procedures. An LC/MS/MS method was developed for the quantitative detection of AAS glucuronides, using a deuterium-labeled steroid glucuronide as the internal standard. LPME, owing to its high specificity, was shown to be better suited than conventional LLE and SPE for the clean-up of urinary AAS glucuronides. The LPME/LC/MS/MS method was fast and reliable, offering acceptable reproducibility and linearity with detection limits in the range 2-20 ng ml(-1) for most of the selected AAS glucuronides. The method was successfully applied to in vitro metabolic studies, and also tested with an authentic forensic urine sample. For a urine matrix the method still has some unsolved problems with specificity, which should be overcome before the method can be reliably used for doping analysis, but still offering additional and complementary data for current GC/MS analyses.     

Sensitive measurement of vinorelbine in dog plasma by liquid chromatography–electrospray ionization tandem mass spectrometry utilizing transitions from double‐charged precursor ions

A sensitive high‐performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for measuring vinorelbine was developed. A 100 µL aliquot of plasma was spiked with deuterium‐labeled internal standard and subjected to solid‐phase extraction using an Oasis HLB μ‐elution plate. Two microliters of the extracted samples was directly injected into LC/MS/MS. Chromatographic separation was achieved on a Capcell Pak C18 UG column (2 × 75 mm) with a gradient elution of methanol (mobile phase B) against 0.05% formic acid in aqueous 10 mm ammonium formate (mobile phase A). The LC flow rate was set to 0.28 mL/min and the gradient (solvent B concentration) was processed from 40 to 90%. In mass spectrometric detection, observation of the reaction from a double‐charged precursor ion [M + 2H]²⁺ (m/z 390) to product ion m/z 122 provided very high sensitivity. The method was validated with a lower limit of detection of 0.2 ng/mL with 0.1 mL of plasma, and the method was used to determine the plasma pharmacokinetics of vinorelbine in dogs. Copyright © 2010 John Wiley & Sons, Ltd.     

A novel approach for sensitivity enhancement in atmospheric pressure chemical ionisation liquid chromatography/mass spectrometry of chlorophylls

Differences in the ionisation efficiency of chlorophylls and their phaeophytin counterparts result in lower sensitivity for atmospheric pressure chemical ionisation mass spectrometric detection of the former. Improvement in the sensitivity of detection of chlorophyll of around an order of magnitude at a concentration of 1 x 10(-6)mol L(-1) has been achieved using post-column addition of methanoic acid during analysis by liquid chromatography/mass spectrometry (LC/MS). The method gives linear response and is a simple strategy to improve sensitivity both for LC/MS and LC/MS/MS without loss of information relating to the precise nature of the tetrapyrrole distributions. Detection levels achieved exceed those obtained by absorbance detection.     

Determination of jasmonic acid in <Emphasis Type="Italic">Lemna minor</Emphasis> (L.) by liquid chromatography with fluorescence detection

A new method is described for the determination of endogenous jasmonic acid (JA) in Lemna minor plant extracts using liquid chromatography (LC) with fluorescence detection. Plant tissues were extracted and derivatised using 9-anthryldiazomethane (ADAM reagent) prepared in situ. Accuracy and precision were improved by using the internal standard dihydrojasmonic acid (dh-JA) for the correction of JA losses during sample preparation steps. Liquid chromatography–mass spectrometry (LC/MS) analysis of ADAM derivatives of JA and dh-JA confirmed that a single molecule of JA and dh-JA was coupled with one molecule of reagent. Derivatives of JA and dh-JA were separated with gradient elution on a C₁₈ reversed-phase column using acetonitrile/water as a mobile phase and detected by a fluorescence detector at excitation and emission wavelengths of 254 and 412 nm, respectively. The detection limits of JA and dh-JA were 2.9 ng mL⁻¹ and 3.7 ng mL⁻¹ per 50-μL injection. The method is reproducible and selective and yields single peaks for each compound regardless of isomer. The specificity and accuracy of the proposed LC/FD method was confirmed by liquid chromatography–TurboIon Spray tandem mass spectrometric (LC/MS/MS) analysis of free JA in Lemna minor samples under multiple reaction monitoring conditions.     

Determination of marker residue of Olaquindox in fish tissue by ultra performance liquid chromatography-tandem mass spectrometry

Methyl-3-quinoxaline-2-carboxylic acid (MQCA) is the last major remaining detectable metabolite of Olaquindox in animal tissue. A rapid, sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the detection and quantification of MQCA in fish tissue using deuterated quinoxaline-2-carboxylic acid (d(4)-QCA) as internal standard. Various parameters affecting sample preparation, LC separation and MS/MS detection were investigated, and the optimal conditions concerned were determined. Fish tissue samples were subject to hydrochloric acid hydrolysis followed by Oasis MAX solid-phase extraction clean-up; analysis was performed using UPLC coupled to electrospray MS/MS. The chromatographic separation was achieved in less than 5 min. The limit of detection and the limit of quantification were 0.1 and 0.25 ng/g, respectively. The average recoveries of MQCA, spiked at levels of 0.25-50.0 ng/g, were from 92.7 to 104.3%. The relative standard deviation values were <6%. The validated method was successfully applied to analyze 60 batch samples collected from the local market.     

Screening for dihydropyridine calcium channel blockers in plasma by automated solid-phase extraction and liquid chromatography/tandem mass spectrometry

A liquid chromatographic/tandem mass spectrometric (LC/MS/MS) screening method was developed for the screening of 11 calcium channel blockers of the 1,4-dihydropyridine type in plasma samples for forensic and clinical cases. Plasma samples were extracted by automated solid-phase extraction. Analysis was performed using a reversed-phase C(18) column, gradient elution and a triple-quadrupole mass spectrometer with TurboIonSpray source in positive mode and multiple reaction monitoring. This method was found to be selective and sensitive for the detection of the target compounds at their therapeutic plasma concentrations.     

Liquid chromatography-electrospray ionisation-mass spectrometry based method for the simultaneous determination of algal and cyanobacterial toxins in phytoplankton from marine waters and lakes followed by tentative structural elucidation of microcystins

A liquid chromatography (LC)-based method with mass spectrometric (MS) detection was developed for simultaneous determination of various algal and cyanobacterial toxins extracted from phytoplankton occurring world-wide in marine waters and lakes. The method enables quantification of saxitoxin, anatoxin-A, domoic acid, nodularin, microcystins, okadaic acid and dinophysistoxin-1 with a single chromatographic run. In addition, the applied chromatographic conditions allow isolation and identification of substances suspected to be "new" microcystins (cyclic peptides) by fraction collection, hydrolysis, derivatisation of resulting free amino acids with the modified chiral Marfey's reagent N-alpha-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA) and enantioselective analysis of the amino acid derivatives by LC-ESI-MS.     

Validity Assessment for the Results of Three Inflammatory Markers in Exhaled Breath Condensate: A Pilot Study

This study was initiated to assess the validity of eicosanoid determination in exhaled breath condensate by immunoassays. The results were compared to those obtained from liquid chromatographic methods with mass spectrometric detection and with the theoretical spiked amount of condensate. Therefore, spiked exhaled breath condensate was prepared achieving samples for comparison of identical aliquots. The three most often analysed inflammatory markers in the literature, i.e. leukotriene B₄, prostaglandin E₂, and 8-isoprostane-prostaglandin F_2α were selected for comparison. Three concentrations of spiked and non-spiked breath condensate matrix were examined. There was an up to 800% overestimation of the eicosanoid quantities revealed by the immunoassay kits used in this study compared to the liquid chromatographic method with mass spectrometric detection which was in line with the nominal amount spiked. Full validation of immunoassay for the determination of inflammatory markers should be performed before further interpretations and consequences will be taken from the absolute values quantified by enzyme-immunoassays. For improving the accuracy of enzyme linked immunoassay (ELISA) results a collaborative work applying ELISA and LC–MS–MS should be performed in order to spotlight the advantages of the ELISA-method.     

Ultrafast liquid chromatography/ultraviolet and liquid chromatography/tandem mass spectrometric analysis

Optimal liquid chromatography/mass spectrometric [LC/MS(/MS)] analysis depends on both the LC selectivity and the electrospray efficiency. Here, we outline a simple and comprehensive LC/MS/MS strategy for the rapid analysis of a wide range of pharmaceutical compounds. To achieve ultrafast LC separation with little sacrifice in peak capacity, one needs to start with a column that provides a good peak capacity at short gradient run times; secondly, it is important to use high flow rates to achieve a good gradient peak capacity. Following this strategy, it was possible to baseline-resolve a mixture (containing acidic, neutral, and basic pharmaceutical analytes) in seconds. By coupling the selectivity provided by fast LC separation with the specificity of MS/MS detection, it is possible to separate and identify a wide range of analytes in 1-min gradient analyses. Also, the impact of mobile phase pH on both the chromatographic selectivity and the MS/MS sensitivity is demonstrated.     

Ionspray mass spectrometry of marine toxins. IV. Determination of diarrhetic shellfish poisoning toxins in mussel tissue by liquid chromatography/mass spectrometry.

An improved liquid chromatographic/mass spectrometric (LC/MS) method utilizing gradient elution and ion-spray ionization is described for the sensitive determination of okadaic acid and dinophysistoxin-1, the principal toxins implicated in cases of diarrhetic shellfish poisoning. The method was used to confirm the presence of both toxins, together with a recently identified isomer of okadaic acid, dinophysistoxin-2, in various samples of cultivated blue mussels (Mytilus edulis) from Canadian and European waters. The method provided a mass detection limit of 0.4 ng for each toxin, thus allowing detection of 40 ng per g of whole mussel tissue (or approximately 10 ng/g if only the digestive glands were used in the assay). Quantitative results obtained by LC/MS were in good agreement with those obtained by derivatization and high-performance liquid chromatography with fluorescence detection.     

Technologies to improve the sensitivity of existing chromatographic methods used for bioanalytical studies

Chromatographic method has long been recognized as the most widely used separation method in bioanalytical research. However, the relatively low sensitivity of existing chromatographic methods remains a significant challenge, as the requirements for experimental procedures become more demanding. This review discusses the main causes for the low sensitivity of chromatographic methods and aims to introduce different technologies for enhancing their sensitivity in the following aspects: (i) different pretreatment methods for improving clean-up efficiency and recovery; (ii) derivatization step for altering the chromatographic behavior of analytes and enhancing MS ionization efficiency; (iii) optimal LC–MS conditions and appropriate separation mechanism; and (iv) applications of other chromatographic methods, including miniaturized LC, 2D-LC, 2D-GC, and supercritical fluid chromatography. Altogether, this review is devoted to summarizing the recent technologies reported in the literature and providing new strategies for the detection of bioanalytes.