Site-specific glycosylation analysis of human apolipoprotein B100 using LC/ESI MS/MS

Human apolipoprotein B100 (apoB100) has 19 potential N-glycosylation sites, and 16 asparagine residues were reported to be occupied by high-mannose type, hybrid type, and monoantennary and biantennary complex type oligosaccharides. In the present study, a site-specific glycosylation analysis of apoB100 was carried out using reversed-phase high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI MS/MS). ApoB100 was reduced, carboxymethylated, and then digested by trypsin or chymotrypsin. The complex mixture of peptides and glycopeptides was subjected to LC/ESI MS/MS, where product ion spectra of the molecular ions were acquired data-dependently. The glycopeptide ions were extracted and confirmed by the presence of carbohydrate-specific fragment ions, such as m/z 204 (HexNAc) and 366 (HexHexNAc), in the product ion spectra. The peptide moiety of glycopeptide was determined by the presence of the b- and y-series ions derived from its amino acid sequence in the product ion spectrum, and the oligosaccharide moiety was deduced from the calculated molecular mass of the oligosaccharide. The heterogeneity of carbohydrate structures at 17 glycosylation sites was determined using this methodology. Our data showed that Asn2212, not previously identified as a site of glycosylation, could be glycosylated. It was also revealed that Asn158, 1341, 1350, 3309, and 3331 were occupied by high-mannose type oligosaccharides, and Asn 956, 1496, 2212, 2752, 2955, 3074, 3197, 3438, 3868, 4210, and 4404 were predominantly occupied by mono- or disialylated oligosaccharides. Asn3384, the nearest N-glycosylation site to the LDL-receptor binding site (amino acids 3359-3369), was occupied by a variety of oligosaccharides, including high-mannose, hybrid, and complex types. These results are useful for understanding the structure of LDL particles and oligosaccharide function in LDL-receptor ligand binding.     

Human plasma quantification of droperidol and ondansetron used in preventing postoperative nausea and vomiting with a LC/ESI/MS/MS method

An analytical method based upon liquid chromatography coupled to ion trap mass spectrometry (MS) detection with electrospray ionization interface has been developed for the simultaneous identification and quantification of droperidol and ondansetron in human plasma. The two drugs were isolated from 0.5 mL of plasma using a basic liquid-liquid extraction with diethyl ether/heptane (90/10, v/v) and tropisetron and haloperidol as internal standards, with satisfactory extraction recoveries. They were separated on a 5-μm C(18) Highpurity column (150 mm×2.1 mm I.D.) maintained at 30°C. The elution was achieved isocratically with a mobile phase of 2 mM HCOONH(4) pH 3.8 buffer/acetonitrile (60/40, v/v) at a flow rate of 200 μL/min. Data were collected either in full-scan MS mode at m/z 100-450 or in full-scan MS-MS mode, selecting the [M+H] (+) ion at m/z=294.0 for ondansetron, m/z=285.2 for tropisetron, m/z=380.0 for droperidol and m/z=376.0 for haloperidol. The most intense daughter ion of ondansetron (m/z=212.0) and droperidol (m/z=194.0) were used for quantification. Retention times for tropisetron, ondansetron, droperidol and haloperidol were 2.50, 2.61, 3.10 and 4.68 min, respectively. Calibration curves were linear for both compounds in the 0.50-500 ng/mL range. The limits of detection and quantification were 0.10 ng/mL and 0.50 ng/mL, respectively. The intra- and inter-assay precisions were lower than 6.4% and intra- and inter-assay recoveries were in the 97.6-101.9% range for the three 3, 30 and 300 ng/mL concentrations. This method allows simultaneous and rapid measurement of droperidol and ondansetron, which are frequently co-administrated for the prevention of postoperative nausea and vomiting.     

Enantioselective LC/MS method for the determination of an antimalarial agent Fenozan B07 in dog plasma

A chiral liquid chromatography/mass spectrometry (LC/MS) bioanalytical procedure has been developed for the analysis of the antimalaric agent Fenozan B07 in dog plasma. Normal-phase chromatography involving a phenylcarbamate derivative of cellulose coated on silica gel as the chiral stationary phase was used to resolve (-)-(S,S)-B07 from (+)-(R,R)-B07. The enantiomers were detected by a mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) interface operated in the negative ion mode. A mass spectrum, characterized by a base peak of m/z 285, was obtained for each enantiomer. The m/z 285 ion was very specific for the analysis of both enantiomers in the plasma. The selected ion monitoring analysis of the plasma samples was therefore performed at m/z 285 for quantitative purposes. The enantiomers were extracted from the plasma in a basic medium and purified by solid-phase extraction using a hydrophilic-lipophilic balanced sorbent. A lower limit of quantification of 2 ng/mL in plasma was achieved for both enantiomers. The quantitative procedure reported in this study was highly specific and sensitive, and was validated according to the FDA guidance on bioanalytical method validation.     

GC/MS and LC/MS Phytochemical Analysis of Vigna unguiculata L. Walp Pod

Vigna unguiculata (L. Walp) or Cowpea pod methanolic extracts phytochemical analysis, total phenolic content (TPC), and secondary metabolite profiling were determined using gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) analysis. GC/MS analysis revealed twenty compounds in the extract, while LC/MS analysis identified twenty-four compounds. GC/MS chromatogram analysis suggested the presence of opioid α-N-Normethadol a major constituent found in methanolic extract and fatty acid esters carotenoid is found second major constituent. LC/MS chromatogram and the mass spectral analysis demonstrated the presence of flavonoids, carotenoids, and alkaloids as major phytochemicals. We investigated the antibacterial, anti-fungal, and anti-oxidant activity of pod methanolic extract. The extract was found equally effective against E. coli, S. pyogenes, and P. aeruginosa with MIC 100 μg/mL similar to the standard Ampicillin (MIC 100 μg/mL). C. albicans were found to be most susceptible to Vign unguiculata pods methanolic extract with a MIC of 250 μg/mL. The pod extract showed significant DPPH scavenging activity (IC₅₀=78.38±0.15) which suggests its antioxidant potential.     

Chemical derivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/MS/MS

An overview is presented of gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), the two major hyphenated techniques employed in metabolic profiling that complement direct 'fingerprinting' methods such as atmospheric pressure ionization (API) quadrupole time-of-flight MS, API Fourier transform MS, and NMR. In GC/MS, the analytes are normally derivatized prior to analysis in order to reduce their polarity and facilitate chromatographic separation. The electron ionization mass spectra obtained are reproducible and suitable for library matching, mass spectral collections being readily available. In LC/MS, derivatization and library matching are at an early stage of development and mini-reviews are provided. Chemical derivatization can dramatically increase the sensitivity and specificity of LC/MS methods for less polar compounds and provides additional structural information. The potential of derivatization for metabolic profiling in LC/MS is demonstrated by the enhanced analysis of plant extracts, including the potential to measure volatile acids such as formic acid, difficult to achieve by GC/MS. The important role of mass spectral library creation and usage in these techniques is discussed and illustrated by examples.     

Biological monitoring of bisphenol A with HLPC/FLD and LC/MS/MS assays

Biological monitoring is a necessary process for risk assessment of endocrine disrupting chemicals (EDCs), particularly, bisphenol A (BPA), in breast milk, because its human risks are not clear yet, and infants, who feed on breast milk, are highly susceptible for EDCs. Concerning biological monitoring of BPA, the HPLC/FLD has been widely used before the LC/MS/MS. However, there was no report, which simultaneously evaluated the two methods in real analyses. Therefore, we analyzed BPA with LC/MS/MS and HPLC/FLD in human breast milk and conducted comparison of two methods in analyzed BPA levels. After establishing optimal condition, e.g. linearity, recovery, reproducibility and free BPA system, we analyzed BPA levels in human breast milk samples (N = 100). The LOQs were similar in the two methods, i.e. 1.8 and 1.3 ng/mL for the HPLC/FLD and LC/MS/MS assays, respectively. There were strong associations between total BPA levels with the two methods (R² = 0.40, p < 0.01), however, only 11% of them were analyzed as similar levels with 15% CVs. In addition, the detection range of BPA was broader in the HPLC method than the LC/MS/MS method. However, the BPA levels in the HPLC/FLD analysis were lower than those in the LC/MS/MS analysis (p < 0.01). Thus, the differences in BPA levels between the two methods may come from mainly over-estimation with the LC/MS/MS method in low BPA samples and some of poor resolution with the HPLC/FLD in high BPA samples.     

Automated identification and quantification of protein phosphorylation sites by LC/MS on a hybrid triple quadrupole linear ion trap mass spectrometer

Complete phosphorylation mapping of protein kinases was successfully undertaken using an automated LC/MS/MS approach. This method uses the direct combination of triple quadrupole and ion trapping capabilities in a hybrid triple quadrupole linear ion trap to selectively identify and sequence phosphorylated peptides. In particular, the use of a precursor ion scan of m/z -79 in negative ion mode followed by an ion trap high resolution scan (an enhanced resolution scan) and a high sensitivity MS/MS scan (enhanced product ion scan) in positive mode is a very effective method for identifying phosphorylation sites in proteins at low femtomole levels. Coupling of this methodology with a stable isotope N-terminal labeling strategy using iTRAQtrade mark reagents enabled phosphorylation mapping and relative protein phosphorylation levels to be determined between the active and inactive forms of the protein kinase MAPKAPK-1 in the same LC/MS run.     

Code developments to improve the efficiency of automated MS/MS spectra interpretation

We report the results of our work to facilitate protein identification using tandem mass spectra and protein sequence databases. We describe a parallel version of SEQUEST (SEQUEST-PVM) that is tolerant toward arithmetic exceptions. The changes we report effectively separate search processes on slave nodes from each other. Therefore, if one of the slave nodes drops out of the cluster due to an error, the rest of the cluster will carry the search process to the end. SEQUEST has been widely used for protein identifications. The modifications made to the code improve its stability and effectiveness in a high-throughput production environment. We evaluate the overhead associated with the parallelization of SEQUEST. A prior version of software to preprocess LC/MS/MS data attempted to differentiate the charge states of ions. Singly charged ions can be accurately identified, but the software was unable to reliably differentiate tandem mass spectra of +2 and +3 charge states. We have designed and implemented a computational approach to narrow charge states of precursor ions from nominal resolution ion-trap tandem mass spectra. The preprocessing code, 2to3, determines the charge state of the precursor ion using its mass-to-charge ratio (m/z) and fragment ions contained in the tandem mass spectrum. For each possible charge state the program calculates the expected fragment ions that account for precursor ion m/z values. If any one of the numbers is less than an empirically determined threshold value then the spectrum corresponding to that charge state is removed. If both numbers are higher than the threshold value then +2 and +3 copies of the spectrum are kept. We present the comparison of results from protein identification experiments with and without using 2 to 3. It is shown that by determining the charge state and eliminating poor quality spectra 2to3 decreases the number of spectral files to be searched without affecting the search results. The decrease reduces computer requirements and researcher efforts for analysis of the results.     

Metabolite profiling of saponins in <Emphasis Type="Italic">Balanites</Emphasis><Emphasis Type="Italic">aegyptiaca</Emphasis> plant tissues using LC (RI)-ESI/MS and MALDI-TOF/MS

Saponins are a major family of secondary metabolites which consist of a sugar moiety glycosidically linked to a hydrophobic aglycone (sapogenin). In recent years the interest in saponins has increased significantly because of their diverse properties as natural detergents and foaming agents, their cardiac, immunostimulating, and anti-cancer activity, as well as other health promoting functions. This study deals with metabolitic analysis of saponins from methanolic extracts of fruit mesocarp (ME), seed kernel (KE) and root (RE) of Balanites aegyptiaca (L.) Del. (desert date) plant grown in Israel using LC (RI)-ESI/MS and MALDI-TOF/MS. The structural assignment was carried out by fragmentation experiments of LC (RI)-ESI/MS and literature data. The study has revealed that, all together, twenty-four furostanol saponins were found in ME, KE and RE. Of these, four saponins are found only in ME, five only in KE and six only in RE. Diosgenin was found to be the sole aglycone in all the saponins. The smallest saponin (MW 740 Da) was found with two sugar units (glucose) and the largest saponin (MW 1678 Da) was found with eight sugar units (5 glucose, 2 rhamnose and 1 xylose) attached to diosgenin. The results suggest that MALDI-TOF/MS with positive ion mode is particularly effective for determining the metabolites of saponins in B. aegyptiaca plant tissues. MALDI-TOF/MS not only verified the results of the LC (RI)-ESI/MS, but also identified additional saponins that are now systematically organized in a database of B. aegyptiaca saponins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

MS/MS profiling of taxoids from the needles of Taxus wallichiana

Ammonium cationisation has been used for taxoid profiling of partially purified methanolic extracts of needles of Taxus wallichiana growing in different regions of the Himalayas (Kashmir, Himachal Pradesh, UP Hills, Darjeeling, Sikkim and Arunachal Pradesh) by electrospray ionisation tandem mass spectrometry (MS/MS). The MS/MS spectra of the [M + NH4]+ or [M + H]+ ions gave structurally diagnostic fragment ions which revealed information about the taxane skeleton as well as the number and nature of the substituents. The rearranged 11(15-->1)-abeo-taxanes showed a characteristic elimination of the hydroxyisopropyl group with an acetoxy/benzoyloxy group from C-9. The identification of the taxoids was achieved by comparison of the MS/MS spectra with those of authentic taxoids or was based on biogenetic grounds. The results were corroborated by liquid chromatography-MS analysis. Out of the 50 taxoids identified, 21 belonged to the rearranged class. The presence of paclitaxel in the samples from four regions was confirmed: the study also revealed the occurrence of several basic taxoids in these samples. MS/MS profiling by electrospray ionisation was shown to be a fast and reliable technique for the analysis of taxoid samples.     

Accelerating high quality bioanalytical LC/MS/MS assays using fused-core columns

High quality, ultra-fast bioanalytical LC/MS/MS methods were developed using short columns packed with fused-core particles and high (1.0–3.0 mL/min) flow rates. For more than two years, at flow rates up to 3.0 mL/min, using 0.33 min non-ballistic gradients, these methods were shown to provide comparable or better performance than slower assays for accuracy, precision, sensitivity, specificity, and ruggedness, and met all criteria required by the bioanalytical regulatory guidance.     

Differentiation of the anomeric configuration and ring form of glucosyl-glycolaldehyde anions in the gas phase by mass spectrometry: isomeric discrimination between m/z 221 anions derived from disaccharides and chemical synthesis of m/z 221 standards

Mass spectrometry of disaccharides in the negative-ion mode frequently generates product anions of m/z 221. With glucose-containing disaccharides, dissociation of isolated m/z 221 product ions in a Paul trap yielded mass spectra that easily differentiated between both anomeric configurations and ring forms of the ions. These ions were shown to be glucosyl-glycolaldehydes through chemical synthesis of their standards. By labeling the reducing carbonyl oxygen of disaccharides with 18O to mass discriminate between monosaccharides, it was established that the m/z 221 ions are comprised solely of an intact nonreducing sugar with a two-carbon aglycon derived from the reducing sugar, regardless of the disaccharide linkage position. This enabled the anomeric configuration and ring form of the ion to be assigned and the location of the ion to the nonreducing side of a glycosidic linkage to be ascertained. Detailed studies of experimental factors necessary for reproducibility in a Paul trap demonstrated that the unique dissociation patterns that discriminate between the isomeric m/z 221 ions could be obtained from month-to-month in conjunction with an internal energy-input calibrant ion that ensures reproducible energy deposition into isolated m/z 221 ions. In addition, MS/MS fragmentation patterns of disaccharide m/z 341 anions in a Paul trap enabled linkage positions to be assigned, as has been previously reported with other types of mass spectrometers.     

Relationship Between Phenolic Content Determined by LC/MS/MS and Antioxidant Capacity and Enzyme Inhibition of Cyclotrichium niveum L.

Cyclotrichium niveum (Boiss.) Manden & Scheng belonging to the Lamiaceae family, which is an endemic species in the eastern Anatolian region of Turkey, has an important place in terms of ethno-botany. The phytochemical composition of the plant, inhibition of acetylcholinesterase (AChE) (which hydrolyzes the neurotransmitter acetylcholine), inhibition of paraoxonase for antiatherosclerotic activity (hPON 1) (which detoxifies organophosphates), and antioxidant capacity were all investigated in this study. Phytochemical content was determined by LC/MS/MS, and enzyme inhibition and antioxidant capacity studies were determined by spectrophotometer. Antioxidant capacity of C. niveum extracts (methanol, hexane, and water) was determined by applying ABTS⋅⁺, DPPH⋅, FRAP, and CUPRAC methods. Both the water and the methanol extracts of the C. niveum exhibited significant inhibition on the AChE (IC₅₀ value for methanol and water extract 0.114±0.14 mg/mL (R2:0.997) and 0.178±0.12 mg/mL (R²: 0.994), respectively). In contrast, the methanol and water extracts of the C. niveum did not exhibit the inhibition effect on hPON 1. The highest activity for ABTS⋅⁺ was 66.53 % in the water extract, and DPPH⋅ was 55.03 % in the methanol extract. In the metal-reducing power assay, the absorbance was 0.168±0.04 for FRAP water extract and 0.621±0.01 for CUPRAC methanol extract. According to LC/MS/MS analyses, hydroxybenzoic acid, salicylic acid, syringic acid, acetohydroxamic acid and luteolin determined in the plant extract. As a consequence, C. niveum which has antioxidant, anti-atherogenic and anti-neurodegenerative properties has the potential to be used as a natural medication instead of synthetic drugs used in Alzheimer's patients.     

Improved validation of peptide MS/MS assignments using spectral intensity prediction

A major limitation in identifying peptides from complex mixtures by shotgun proteomics is the ability of search programs to accurately assign peptide sequences using mass spectrometric fragmentation spectra (MS/MS spectra). Manual analysis is used to assess borderline identifications; however, it is error-prone and time-consuming, and criteria for acceptance or rejection are not well defined. Here we report a Manual Analysis Emulator (MAE) program that evaluates results from search programs by implementing two commonly used criteria: 1) consistency of fragment ion intensities with predicted gas phase chemistry and 2) whether a high proportion of the ion intensity (proportion of ion current (PIC)) in the MS/MS spectra can be derived from the peptide sequence. To evaluate chemical plausibility, MAE utilizes similarity (Sim) scoring against theoretical spectra simulated by MassAnalyzer software (Zhang, Z. (2004) Prediction of low-energy collision-induced dissociation spectra of peptides. Anal. Chem. 76, 3908-3922) using known gas phase chemical mechanisms. The results show that Sim scores provide significantly greater discrimination between correct and incorrect search results than achieved by Sequest XCorr scoring or Mascot Mowse scoring, allowing reliable automated validation of borderline cases. To evaluate PIC, MAE simplifies the DTA text files summarizing the MS/MS spectra and applies heuristic rules to classify the fragment ions. MAE output also provides data mining functions, which are illustrated by using PIC to identify spectral chimeras, where two or more peptide ions were sequenced together, as well as cases where fragmentation chemistry is not well predicted.     

Quantitative proteome analysis using differential stable isotopic labeling and microbore LC-MALDI MS and MS/MS

We demonstrate an approach for global quantitative analysis of protein mixtures using differential stable isotopic labeling of the enzyme-digested peptides combined with microbore liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Microbore LC provides higher sample loading, compared to capillary LC, which facilitates the quantification of low abundance proteins in protein mixtures. In this work, microbore LC is combined with MALDI MS via a heated droplet interface. The compatibilities of two global peptide labeling methods (i.e., esterification to carboxylic groups and dimethylation to amine groups of peptides) with this LC-MALDI technique are evaluated. Using a quadrupole-time-of-flight mass spectrometer, MALDI spectra of the peptides in individual sample spots are obtained to determine the abundance ratio among pairs of differential isotopically labeled peptides. MS/MS spectra are subsequently obtained from the peptide pairs showing significant abundance differences to determine the sequences of selected peptides for protein identification. The peptide sequences determined from MS/MS database search are confirmed by using the overlaid fragment ion spectra generated from a pair of differentially labeled peptides. The effectiveness of this microbore LC-MALDI approach is demonstrated in the quantification and identification of peptides from a mixture of standard proteins as well as E. coli whole cell extract of known relative concentrations. It is shown that this approach provides a facile and economical means of comparing relative protein abundances from two proteome samples.     

Characterization of betaines using electrospray MS/MS

Betaines are an important class of naturally occurring compounds that function as compatible solutes or osmoprotectants. Because of the permanent positive charge on the quaternary ammonium moiety, mass spectrometric analysis has been approached by desorption methods, including fast atom bombardment and plasma desorption mass spectrometry. Here we show that electrospray ionization MS gives comparable results to plasma desorption MS for a range of authentic betaine standards and betaines purified from plant extracts by ion exchange chromatography. A distinct advantage of electrospray ionization MS over plasma desorption MS is the capability of obtaining product ion spectra via MS/MS of selected parent ions, and hence structural information to discriminate between ions of identical mass.     

Postexperiment monoisotopic mass filtering and refinement (PE-MMR) of tandem mass spectrometric data increases accuracy of peptide identification in LC/MS/MS

Methods for treating MS/MS data to achieve accurate peptide identification are currently the subject of much research activity. In this study we describe a new method for filtering MS/MS data and refining precursor masses that provides highly accurate analyses of massive sets of proteomics data. This method, coined "postexperiment monoisotopic mass filtering and refinement" (PE-MMR), consists of several data processing steps: 1) generation of lists of all monoisotopic masses observed in a whole LC/MS experiment, 2) clusterization of monoisotopic masses of a peptide into unique mass classes (UMCs) based on their masses and LC elution times, 3) matching the precursor masses of the MS/MS data to a representative mass of a UMC, and 4) filtration of the MS/MS data based on the presence of corresponding monoisotopic masses and refinement of the precursor ion masses by the UMC mass. PE-MMR increases the throughput of proteomics data analysis, by efficiently removing "garbage" MS/MS data prior to database searching, and improves the mass measurement accuracies (i.e. 0.05 +/- 1.49 ppm for yeast data (from 4.46 +/- 2.81 ppm) and 0.03 +/- 3.41 ppm for glycopeptide data (from 4.8 +/- 7.4 ppm)) for an increased number of identified peptides. In proteomics analyses of glycopeptide-enriched samples, PE-MMR processing greatly reduces the degree of false glycopeptide identification by correctly assigning the monoisotopic masses for the precursor ions prior to database searching. By applying this technique to analyses of proteome samples of varying complexities, we demonstrate herein that PE-MMR is an effective and accurate method for treating massive sets of proteomics data.     

Method for therapeutic drug monitoring of azole antifungal drugs in human serum using LC/MS/MS

Fungal infections occur in immunocompromised patients. Azole antifungal agents are used for the prophylaxis and treatment of these infections. The interest in therapeutic drug monitoring azole agents has increased over the last few years. Inter- and intra-patient variability of pharmacokinetics, drug–drug interactions, serum concentration related toxicity and success of therapy has stressed the need of frequently therapeutic drug monitoring of the drugs, belonging to the group of azoles. Therefore a simple, rapid and flexible method of analysis is required. This method is based on the precipitation of proteins in human serum with LC/MS/MS detection. Validation was performed according to the guidelines for bioanalytical method validation of the food and drug administration agency. The four most used azole drugs can be detected in human serum within the clinical relevant serum levels with good accuracy and reproducibility at the limit of quantification. Intra- and inter-day validation demonstrated good accuracy and reproducibility. A rapid, sensitive and flexible LC/MS/MS method has been developed and validated to measure voriconazole (VRZ), fluconazole (FLZ), itraconazole (ITZ) and posaconazole (PSZ) in human serum. This new method is suitable for clinical pharmacokinetic studies and routine monitoring in daily practice.     

The combination effects of phenolic compounds and fluconazole on the formation of ergosterol in Candida albicans determined by high-performance liquid chromatography/tandem mass spectrometry

A liquid chromatography/tandem mass spectrometry (LC/MS) with atmospheric pressure chemical ionization (APCI) for the quantification of ergosterol, lanosterol, and squalene was developed to evaluate the combination effects of phenolic compounds with fluconazole on ergosterol biosynthesis in Candida albicans. The three analytes were separated by a column of C18 and were quantified without interference with each other using positive mode tandem mass spectrometry (MS/MS). Molecular ions of ergosterol and lanosterol were detected as the [M+H-H2O]+ ion species at m/z 380 and 410, whereas squalene appeared as the [M+H]+ ion species at m/z 412. On fragmentation of ergosterol, lanosterol, and squalene, the product ions at m/z 69, 149, and 109, respectively, were present as major fragments. These product ions were used for the quantification of them in multiple reaction monitoring acquisition mode. The relationship between signal intensity and the analytes' concentration was linear over the concentration range of 0.05-10 microg/ml. Following the treatment of C. albicans with fluconazole in combination with albicanyl caffeate, resveratrol, and 3,4'-difluorostilbene, respectively, the content of ergosterol in both the sensitive and resistant C. albicans showed depletion, whereas the squalene showed accumulation especially in the sensitive isolates determined with the method developed.