Monitoring phospholipids for assessment of ion enhancement and ion suppression in ESI and APCI LC/MS/MS for chlorpheniramine in human plasma and the importance of multiple source matrix effect evaluations

Biological matrix effects are a source of significant errors in both electrospray (ESI) and atmospheric pressure chemical ionization (APCI) LC/MS. Glycerophosphocholines (GPChos) and 2-lyso-glycerophosphocholines (2-lyso GPChos) are known to fragment to form ions at m/z 184 and m/z 104, respectively. Phospholipids were used as markers to evaluate matrix effects resulting in both ion suppression and enhancement using ESI and APCI modes in the determination of chlorpheniramine in human plasma. Results revealed that GPChos and 2-lyso GPChos demonstrated very low ionization efficiency in the APCI mode, post-column infusion experiments were performed to confirm that suppression and enhancement matrix ionization effects coincided with the elution profiles of the phospholipids. The mean matrix effect for chlorpheniramine using APCI was 75% less than the mean matrix effect in ESI, making APCI the ionization method of choice initially even though the absolute response was lower than in the ESI mode. The resulting APCI method showed acceptable results according to the FDA guidelines; however, a multiple source relative matrix effects study demonstrated variability. It was concluded that an absolute matrix effects study in one source of biological fluid may be not sufficient to ensure the validity of the method in various sources of matrix. In order to obviate the multiple matrix source variability, we employed an isotopically labeled internal standard for quantification of chlorpheniramine in the ESI mode. An additional validation was completed with the use of chlorpheniramine-d(6) as the internal standard. This method met all acceptance criteria according to the FDA guidelines, and the relative matrix affects study was successful.     

Mass spectrometry determination of endonuclear phospholipid composition and dynamics

Mammalian cell lipid analyses using tandem electrospray ionization mass spectrometry, in conjunction with stable isotope labeling, permit unparalleled access to membrane phospholipid molecular species compositions and turnover. Lipidomic data from isolable compartments of lipid second messenger generation, such as membrane-free nuclei, can provide dynamic insights into the topology of phospholipid turnover. For example, ESI-MS/MS precursor scans of characteristic phosphocholine m/z 184(+) fragments reveal a highly saturated endonuclear phosphatidylcholine pool with homeostatic maintenance properties. A spatially distinct CDPcholine pathway yields, within minutes of choline-d(9) labeling, unsaturated endonuclear phosphatidylcholines progressively remodeled to more saturated species evidenced by tracking the deuteriated headgroup through precursor scans of phosphocholine-d(9) (m/z 193(+) fragment). Among the other endonuclear phospholipids, diacyl phosphatidylethanolamines (neutral loss of m/z 141(+)) are also highly saturated compared with those of whole cell whereas, phophatidylinositols (precursor scans of m/z 241(-) fragment) are essentially identical in nuclei and whole cells. Moreover, the pattern of myo-inositol-d(6) acquisition into endonuclear phosphatidylinositol (precursor scans of m/z 247(-) fragment) is inconsistent with compartment-specific synthesis. Endonuclear sphingomyelins (seen in precursor scans of m/z 184(+) and confirmed from precursor scans of m/z 168(-) fragments) are enriched but similar in composition to whole cell species whereas endonuclear phosphatidylserines (neutral loss of m/z 87(-)) are more saturated than their whole cell counterparts. The focus of described methodologies emphasize their value in probing the compositions and dynamics of endonuclear phospholipids, but in principle may be extended to exploration of other isolable compartments including ER or plasma membranes.     

Liquid chromatography-mass spectrometry/mass spectrometry method development for drug metabolism studies: Examining lipid matrix ionization effects in plasma

Glycerophosphocholines (GPCho's) are known to cause liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) matrix ionization effects during the analysis of biological samples (i.e. blood, plasma). We have developed a convenient new method, which we refer to as "in-source multiple reaction monitoring" (IS-MRM), for detecting GPCho's during LC-MS/MS method development. The approach uses high energy in-source collisionally induced dissociation (CID) to yield trimethylammonium-ethyl phosphate ions (m/z 184), which are formed from mono- and disubstituted GPCho's. The resulting ion is selected by the first quadrupole (Q1), passed through the collision cell (Q2) in the presence of collision gas at low energy to minimize fragmentation, and m/z 184 selected by the third quadrupole. This approach can be combined with standard multiple reaction monitoring (MRM) transitions with little compromise in sensitivity during method development and sample analysis. Hence, this approach was used to probe ionization matrix effects in plasma samples. The resulting information was employed to develop LC-MS/MS analyses for drugs and their metabolites with cycle times less than 5 min.     

Investigation of endogenous blood plasma phospholipids, cholesterol and glycerides that contribute to matrix effects in bioanalysis by liquid chromatography/mass spectrometry

Matrix effects caused by compounds endogenous to the biological sample are a primary challenge in quantitative LC/MS/MS bioanalysis. Many approaches have been developed to minimize matrix effects such as optimization of sample extraction procedures and use of isotopically labeled internal standards. Unexpected matrix components may still remain undetected, however, because of the selective mass transitions monitored during MS/MS analysis. Glycerophosphocholines are the major phospholipids in plasma that have been widely shown to cause significant matrix effects on electrospray ionization efficiencies for target analytes. The purpose of this work was to investigate potential matrix effects resulting from different endogenous lipid classes, including phospholipids, acylglycerols and cholesterols, in order to establish a library for the relative presence of these components in biological sample extracts obtained by commonly used sample preparation techniques. Thirteen compounds were selected which were representatives of eight phospholipids classes, mono, di, triacylglycerols, cholesterol and cholesterol esters. Post-column infusion experiments were carried out to compare relative ion suppression effects of these compounds. Chlorpheniramine and loratadine were selected as model test analytes. A Concentration Normalized Suppression Factor (%CNSF) was defined to allow comparison of ion suppression effects resulting from different endogenous lipids according to their typical concentrations in human plasma and erythrocytes. A simple LC/MS/MS method was developed to monitor these endogenous components in sample extracts and their extraction recoveries from a plasma pool were compared using protein precipitation, liquid-liquid extraction, supported-liquid extraction, solid phase extraction and Hybrid SPE-precipitation methods. Endogenous lipid components other than GPChos, such as cholesterols and triacylglycerols, may result in significant matrix effects and should be monitored during method development. No single extraction procedure was efficient in removing all of the various lipid components. Use of the results presented here, along with a consideration of analyte chemical structure, the type of matrix and the type of sample preparation procedure, may help a bioanalytical scientist to better anticipate and minimize matrix effects in developing LC/MS/MS-based methods.     

Ion suppression effects in liquid chromatography-electrospray-ionisation transport-region collision induced dissociation mass spectrometry with different serum extraction methods for systematic toxicological analysis with mass spectra libraries

Ion suppression effects during electrospray-ionsation mass spectrometry (ESI-MS) caused by different sample preparation procedures for serum were investigated. This topic is of importance for systematic toxicological analysis for which LC-ESI-MS has been developed with transport-region collision-induced dissociation (ECI-CID) and mass spectra library searching. With continuous postcolumn infusion of two test compounds-codeine and glafenine-the ion suppression effects of extracted biological matrix obtained after a standard liquid-liquid extraction, a mixed-mode solid-phase extraction (SPE) method, a protein precipitation method and a combination of precipitation with polymer-based mixed-mode SPE have been investigated. Extracted ion chromatograms of codeine ([M+H](+), m/z 300) and glafenine ([M-H](-), m/z 371) were used for monitoring ion suppression. Severe ion suppression effects for codeine and glafenine were detected in positive and in negative ionisation modes, respectively, in the LC-front peak after serum clean-up with SPE (acid/neutral fraction) and protein precipitation as well as with protein precipitation combined with SPE. Less ion suppression of codeine in positive mode was found with liquid-liquid extraction of serum samples. No ion suppression was detected with the second fraction of the mixed-mode SPE (using RP-C(8) and cation-exchange phase) in both ionisation modes. All suppression effects were caused by polar and unretained matrix components, which were present after extraction and/or protein precipitation. However, no specific ion suppression was seen after elution of the polar LC-front throughout the whole gradient. It could be demonstrated, that ion suppression is not generally present at any retention time when using reversed-phase HPLC with rather long gradient programs, but may play an important role in case of high-throughput LC-MS analysis, when the analyte is not separated from the LC-front, or in flow injection analysis without chromatographic separation.     

Targeted metabolomic analysis of Escherichia coli by desorption electrospray ionization and extractive electrospray ionization mass spectrometry

Desorption electrospray ionization (DESI) was utilized to monitor the presence of targeted central carbon metabolites within bacterial cell extracts and the quench supernatant of Escherichia coli. The targeted metabolites were identified through tandem mass spectrometry (MS/MS) product ion scans using collision-induced dissociation in the negative ion mode. Picogram detection limits were achieved for a majority of the metabolites during MS/MS analysis of standard metabolite solutions. In a [U-(13)C]glucose pulse experiment, where uniformly labeled glucose was fed to E. coli, the corresponding fragment ions from labeled metabolites in extracts were generally observed. There was evidence of matrix effects including moderate suppression by other metabolites within the spectra of the labeled and unlabeled extracts. To improve the specificity and sensitivity of detection, optimized in situ ambient chemical reactions using DESI and extractive electrospray ionization (EESI) were carried out for targeted compounds. This study provides the first indication of the potential to perform in situ targeted metabolomics of a bacterial sample via ambient ionization mass spectrometry.     

High-performance liquid chromatography of phospholipids with UV detection: optimization of separations on silica

Chromatography of phospholipids was performed on silica columns with detection by absorbance at 205 nm using mixtures of hexane—isopropanol—water in which the role of water and isopropanol in elution was investigated. One system was developed which provided adequate separation of most major phospholipid species. However, lipids with several ionizable groups were not well separated and gave multiple broad peaks. A second system was developed utilizing sulfuric acid for ion suppression. The behavior of phospholipids in this system was found to be dependent on the presence of quaternary ammonium, amino, or hydroxyl groups. Except for plasmalogen, phospholipids were recovered intact. This system was optimized to provide baseline resolution of essentially all phospholipid species commonly found in mammalian tissues.     

A method for profiling gangliosides in animal tissues using electrospray ionization-tandem mass spectrometry

Gangliosides are critical in many functions of mammalian cells but present as a minor lipid component with many molecular species of subtle differences. Conventional strategies for profiling gangliosides suffer from poor reproducibility, low sensitivity, and low-throughput capacity. Prior separation of gangliosides by thin-layer chromatography and/or high-performance liquid chromatography not only was laborious and tedious but also could introduce uneven losses of molecular species. We developed a new strategy of using electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to profile gangliosides with high-throughput potential. This strategy involves three new findings: (i) collision-induced fragmentation of gangliosides gave rise to a common ion of m/z 290, a derivative of N-acetylneuraminic acid; (ii) phospholipids exert a profound suppression of ganglioside detection in ESI-MS/MS to prevent a direct detection in total cellular lipid extracts; and (iii) enrichment of gangliosides in the aqueous phase from total cellular lipid extracts eliminates the damping effect of phospholipids and permits direct precursor scan.     

Development of a reverse-phase liquid chromatography electrospray ionization mass spectrometry method for lipidomics, improving detection of phosphatidic acid and phosphatidylserine

In the studies of lipid metabolomics, liquid chromatography electrospray ionization mass spectrometry (LC/MS) is a robust and popular technique. Although effective reverse-phase LC methods enabling the separation of phospholipid molecular species have been developed, there are still problems with the separation of phosphatidic acid (PA) and phosphatidylserine (PS). These acidic phospholipids often elute as extensively broad peaks, causing inferior separation, detection, and quantification-a severe limitation of the method. In this study, we have developed reverse-phase LC conditions that reduce the undesired peak tailings in the elution profiles of both PA and PS, by using a starting mobile phase containing a low concentration of phosphoric acid (5 microM) and a high percentage of water (40%). Our method sensitively analyzes PA, PS, and their lysoforms, as well as the other phospholipids within a biological sample, in a single chromatographic step by an LC/MS method and, thus, is suitable for lipidomics.     

Phospholipid molecular species distribution of some medically important Candida species analysed by fast atom bombardment mass spectroscopy

The aim of this study was to obtain detailed information on phospholipids (PL) of the medically important Candida species and to determine their possible chemotaxonomic significance. Lipids were extracted from 22 strains representing 8 Candida species and their PL molecular species distributions were determined by Fast Atom Bombardment Mass Spectroscopy (FAB MS) in negative ion mode. Fifteen major lower mass peaks (m/z 221 to 289) were attributable to the expected presence of carboxylate anions and 24 major higher mass peaks (m/z 557 to 837) were attributable to phospholipid anions. Major carboxylate peaks were of the following m/z and identities : 253, C16:1; 255, C16:0; 277, C18:3; 279, C18:2; 281, C18:1; and 283, C18:0. The most abundant peaks consistent with the presence of phospholipid molecular species anions include those of m/z 673, 743, 833, 834 and 836 tentatively identified as phosphatidic acid (PA) (34:1), phosphatidylglycerol (PG) (34:3), phosphtidylinositol (PI) (34:2) and two unknown molecular species. This profile is diagnostic for the genus Candida. Quantitative differences were observed between different Candida species. Thus, polar lipid molecular species distribution in Candida spp. has chemotaxonomic significance, especially so in the case of carboxylate anions.     

Site localization of sialyl Lewis(x) antigen on alpha1-acid glycoprotein by high performance liquid chromatography-electrospray mass spectrometry

A simple, fast and sensitive method was developed to verify the presence of the sialyl Lewis(x) antigen on an N-linked glycoprotein. High performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI/MS) was used to identify which of the five N-linked glycosylation sites of human plasma alpha1-acid-glycoprotein (orosomucoid, OMD) contain the sialyl Lewis(x) antigen. OMD was digested with proteolytic enzymes and analyzed by reversed phase chromatography coupled with on-line ESI/MS. A tandem mass spectrometry experiment was designed to detect the presence of the sialyl Lewis(x) antigen based on the observation of an 803 mass to charge ratio ( m/z ) ion produced in the intermediate pressure region of the ESI interface. The ESI/MS signal at m/z 803 is consistent with an oxonium ion for a glycan structure containing NeuAc, Gal, GlcNAc, and Fuc. The identity of the m/z 803 ion was confirmed by ESI/MS/MS analysis of the m/z 803 fragment ion and comparison with a sialyl Lewis(x) standard. The stereochemistry and linkage positions were assigned using previous NMR analysis but could be determined with permethylation analysis if necessary. The analysis of OMD gave a pattern showing signal for the sialyl Lewis(x) antigen coeluting with each of the five N-linked glycopeptides. The ability to monitor sialyl Lewis(x) expression at each of the five sites is of interest in the study of OMD's role in inflammatory diseases.      

High-performance liquid chromatography for the determination of 3-n-butylphthalide in rat plasma by tandem quadrupole mass spectrometry: application to a pharmacokinetic study

A rapid, sensitive and specific high performance liquid chromatography-electrospray ionization tandem quadrupole mass spectrometry (HPLC-MS/MS) method was developed and validated for the determination of 3-n-butylphthalide in rat plasma. Following protein precipitation with acetonitrile, 3-n-butylphthalide and glipizide (internal standard, I.S.) were separated using a gradient elution program on a C18 column and detected by mass spectrometry in positive ion mode with the multiple reaction monitoring (MRM) mode using the respective precursor to product ion combinations of m/z 191/145 for 3-n-butylphthalide and m/z 446/321 for glipizide, respectively. The total chromatographic running time was 2.5 min. The method was linear over the concentration range of 11.14-3480.00 ng/mL, using as little as 100 microL plasma. The lower limit of quantification (LLOQ) was 5.57 ng/mL. Finally, the method was successfully used to support a preclinical pharmacokinetic study of 3-n-butylphthalide in rats following intravenous administration.     

Characterization of astaxanthin esters in Haematococcus pluvialis by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

After first being analyzed by HPLC, 4 free carotenoids, 15 astaxanthin monoesters, 12 astaxanthin diesters, and 3 astacin monoesters in Haematococcus pluvialis were identified by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-(APCI)MS). Identification of each compound was based on the characteristic fragment ions of the positive ion mode, negative ion mode, and MS(2). Astaxanthin esters were identified based on the loss of one or two fatty acids. In a positive ion mode, astaxanthin monoesters had characteristic fragment ions at m/z 597 [M+H-fatty acid](+) and m/z 579 and 561 that resulted from a continuous loss of water. The relative intensity of m/z 579 in MS(2) amounted to more than 80% of that of the molecular ion. In astaxanthin diesters, the intensity of m/z 561 occasionally was equal to that of m/z 579, but in general the former, amounting to 50 to 60% or more of the molecular ion, was stronger than the latter, which decreased to 20 to 30% of the molecular ion. In addition, a set of compounds with maximum absorbance at 400 nm, detected by high-performance liquid chromatography-diode array detector (HPLC-DAD), had strong characteristic fragment ions at m/z 871 and 593 in the positive ion mode MS(2). They were presumed to be linolenic acid or an isomer of omega-6-gamma-linolenic acid esters of astacin.     

Simultaneous quantitation of dexamethasone palmitate and dexamethasone in human plasma by liquid chromatography/tandem mass spectrometry

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for simultaneous quantitation of dexamethasone palmitate and dexamethasone in human plasma was developed. After sample preparation by protein precipitation and liquid-liquid extraction, the analytes and internal standard (IS) were separated on a Venusil XBP-C8 column using gradient elution. Multiple reaction monitoring of dexamethasone palmitate, dexamethasone and IS used the precursor to product ion transitions at m/z 631.8-->373.1, m/z 393.2-->147.1 and m/z 264.2-->58.1, respectively. The method was linear over the ranges 1.5-1000ng/mL for dexamethasone palmitate and 2.5-250ng/mL for dexamethasone with intra- and inter-day precisions of <10% and accuracies of 100+/-7%. The assay was applied to a clinical pharmacokinetic study involving the injection of dexamethasone palmitate to healthy volunteers.     

An approach to enhancing coverage of the urinary metabonome using liquid chromatography-ion mobility-mass spectrometry

The potential of drift tube ion mobility (IM) spectrometry in combination with high performance liquid chromatography (LC) and mass spectrometry (MS) for the metabonomic analysis of rat urine is reported. The combined LC-IM-MS approach using quadrupole/time-of-flight mass spectrometry with electrospray ionisation, uses gas-phase analyte characterisation based on both mass-to-charge (m/z) ratio and relative gas-phase mobility (drift time) following LC separation. The technique allowed the acquisition of nested data sets, with mass spectra acquired at regular intervals (65 micros) during each IMS separation (approximately 13 ms) and several IMS spectra acquired during the elution of a single LC peak, without increasing the overall analysis time compared to LC-MS. Preliminary results indicate that spectral quality is improved when using LC-IM-MS, compared to direct injection IM-MS, for which significant ion suppression effects were observed in the electrospray ion source. The use of reversed-phase LC employing fast gradient elution reduced sample preparation to a minimum, whilst maintaining the potential for high throughput analysis. Data mining allowed information on specific analytes to be extracted from the complex metabonomic data set. LC-IM-MS based approaches may have a useful role in metabonomic analyses by introducing an additional discriminatory dimension of ion mobility (drift time).     

Determination of octreotide and assessment of matrix effects in human plasma using ultra high performance liquid chromatography-tandem mass spectrometry

A selective UHPLC-MS/MS method for determination of the therapeutic peptide octreotide in human plasma was developed and validated. This assay used a UHPLC C(18) column with 1.7 μm particle size for efficient separation and an ion-exchange SPE for selective extraction. Octreotide and its labeled internal standard, [(13)C(6)Phe(3)] octreotide, were extracted from human plasma using a simple Oasis? WCX μElution SPE method and analyzed with a total chromatographic run time of 7.5 min. Matrix effects were studied during method development by direct monitoring of representative phospholipids. On-line removal of phospholipids using column switching and pre-column back-flushing was carried out to trap and remove any residual phospholipid matrix interferences. The UHPLC column provided baseline separation between the analyte and matrix peaks. The chromatographic conditions yielded optimal retention and excellent peak shape for both the analyte and internal standard. The assay was linear in the concentration range of 0.025-25.0 ng/ml, inter- and intra-assay precision and accuracy were within 6.1% and ±1.93%, respectively. Recovery was ～73%. Post-extraction addition experiments showed that matrix effects were less than 4%. This method for octreotide in human plasma has been validated and utilized to support of clinical pharmacokinetic studies.     

Simultaneous determination of asperosaponin VI and its active metabolite hederagenin in rat plasma by liquid chromatography-tandem mass spectrometry with positive/negative ion-switching electrospray ionization and its application in pharmacokinetic study

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method operated in the positive/negative electrospray ionization (ESI) switching mode has been developed and validated for the simultaneous determination of asperosaponin VI and its active metabolite hederagenin in rat plasma. After addition of internal standards diazepam (for asperosaponin VI) and glycyrrhetic acid (for hederagenin), the plasma sample was deproteinized with acetonitrile, and separated on a reversed phase C18 column with a mobile phase of methanol (solvent A)-0.05% glacial acetic acid containing 10 mM ammonium acetate and 30 μM sodium acetate (solvent B) using gradient elution. The detection of target compounds was done in multiple reaction monitoring (MRM) mode using a tandem mass spectrometry equipped with positive/negative ion-switching ESI source. At the first segment, the MRM detection was operated in the positive ESI mode using the transitions of m/z 951.5 ([M+Na](+))→347.1 for asperosaponin VI and m/z 285.1 ([M+H](+))→193.1 for diazepam for 4 min, then switched to the negative ESI mode using the transitions of m/z 471.3 ([M-H](-))→471.3 for hederagenin and m/z 469.4 ([M-H](-))→425.4 for glycyrrhetic acid, respectively. The sodiated molecular ion [M+Na](+) at m/z 951.5 was selected as the precursor ion for asperosaponin VI, since it provided better sensitivity compared to the deprotonated and protonated molecular ions. Sodium acetate was added to the mobile phase to make sure that abundant amount of the sodiated molecular ion of asperosaponin VI could be produced, and more stable and intensive mass response of the product ion could be obtained. For the detection of hederagenin, since all of the mass responses of the fragment ions were very weak, the deprotonated molecular ion [M-H](-)m/z 471.3 was employed as both the precursor ion and the product ion. But the collision energy was still used for the MRM, in order to eliminate the influences induced by the interference substances from the rat plasma. The validated method was successfully applied to study the pharmacokinetics of asperosaponin VI and its active metabolite hederagenin in rat plasma after oral administration of asperosaponin VI at a dose of 90 mg/kg.     

Negative ion fast atom bombardment-tandem mass spectrometry for structural analysis of isoprenoid diphosphates

Applicability of negative ion fast atom bombardment (FAB)-tandem mass spectrometry (MS/MS) was examined in trace mixture analyses and structural assignments of some isoprenoid diphosphates. Negative ion FAB-MS spectra using a glycerol matrix of these isoprenoid diphosphates showed predominantly molecular ions (M-H)- together with fragment ions at m/z 177 (H3P2O7)-, 176 (H2P2O7)-, 159 (HP2O6)-, and 79 (PO3)- which were characteristic of the diphosphate ester moiety. The molecular ions did not overlap with peaks arising from any impurities even when crude sample such as butanol extracts from enzymatic reaction mixtures were directly analyzed without any purification. Moreover, collisionally activated dissociation spectra of the molecular ion showed many structurally significant fragment ions which enabled us to elucidate the structures of such irregular alkyl chain moieties as those having a homoisoprenoid skeleton or substituted structures. These studies indicate that negative ion FAB-MS/MS is a simple and useful technique for trace mixture analysis and structure elucidation of isoprenoid diphosphates.     

Liquid ionization mass spectrometry of phospholipids

Several phosphatidylcholines (PC) and a phosphatidylethanolamine (PE) were subjected to liquid ionization (LI) mass spectrometry, in which a sample is ionized through energy transfer from metastable argon atoms under atmospheric pressure. Commercially available and synthesized, saturated or unsaturated fatty acid containing phospholipids and their mixtures were studied. A sample either as a concentrated chloroform-methanol solution or with glycerol (matrix) gave characteristic peaks such as MH+ and four fragment ions. One of the fragment ions (e.g., m/z 551 of PC 16:0, 16:0) containing both fatty acid residues has been commonly observed with other ionization methods such as CI, FD, and FAB, but the other fragment ions have not been observed in other mass spectra with one exception on desorption CI. Ions b and d (e.g., m/z 464 and 328, respectively, for PC 16:0, 16:0) contain one fatty acyl residue and the other ion containing the phosphorylcholine moiety appears at m/z 196 for PC. Thus the masses of the MH+ ion and these fragment ions provide useful structural information even in the case of a mixture. The ion b (e.g., m/z 488 of PC 18:0, 18:2) observed during an early period of heating was formed mainly by the loss of one acyl group at sn-1 of the glycerol backbone and thus may be used to differentiate the positional specificities of the constituent fatty acids. The temperature of the sample, however, should be controlled precisely, because it has a significant effect on the mass spectrum. The present method (LI) also provided useful information for a mixture of PC and PE.     

Analysis of acetylcholine from extracellular fluid in brain by in vivo microdialysis and LC-ESI-MS/MS with the stable isotope-labeled internal standard

Acetylcholine (ACh) associated with Alzheimer's and Parkinson's disease is the major neurotransmitter in vertebrates. In support of clinical studies on the mechanism of the illnesses and development of medicines for these diseases, the LC-ESI-MS/MS method was developed and validated for the direct quantification of ACh in dialysate samples with acetylcholine-D(9) bromide (IS) as the isotope-labeled internal standard. The analytes were separated on the Waters Hilics C(18) Column (2.1 mm×100 mm, 3 μm) on LC with mobile phase ultrapure water-200 mM ammonium formate (pH 3.04)-acetonitrile (30:5:65, vol/vol/vol) at a flow rate of 300 μL/min, and monitored with a fragment ion of m/z 87 formed from a molecular ion of m/z 146 for ACh and that of m/z 87 from m/z 155 for IS during multiple reaction monitoring (MRM) positive ion mode. The lower limit of quantitation (LLOQ) of ACh was lower than 0.1 nmol/L in dialysate samples, equivalent to 0.2fmol injected on-column. The developed method could be utilized in the analysis of ACh in dialysate samples and these results were in good agreement with the gradient elution study.