Alkylacyl glycerophosphoinositol in human and bovine erythrocytes. Molecular species composition and comparison with glycosyl-inositolphospholipid anchors of erythrocyte acetylcholinesterases.

Glycosyl-inositolphospholipid (glycosyl-PtdIns) anchors of proteins in mammalian cells which have been analyzed so far are exclusively of the alkylacyl type. However, little is known about the putative precursor of glycosyl-PtdIns, the alkylacyl derivative of glycerophosphoinositol (GroPIns), in these cells since it is generally believed that cellular GroPIns consists of diacyl-type molecular species only. In this report, we describe the isolation and identification of alkylacyl GroPIns molecular species in both human and bovine erythrocytes, and compare it with the molecular species compositions of the glycosyl-PtdIns anchors of human and bovine erythrocyte acetylcholinesterase. Diradyl GroPIns was isolated from lipid extracts of ghost membranes and treated with phospholipase C. Diradylglycerols of the glycosyl-PtdIns anchors of affinity-purified human and bovine erythrocyte acetylcholinesterase were generated by sequential treatment with glycoprotein phospholipase D and acidic phosphatase and by PtdIns-specific phospholipase C, respectively. Diradylglycerols were subsequently converted into benzoate derivatives and separated into diacyl, alkylacyl, and alkenylacylglycerol subclasses. The molecular species compositions were quantitated and determined by combined HPLC/mass spectrometry. We found that human and bovine erythrocyte membrane diradyl GroPIns consist of 1.5-4.8% alkylacyl GroPIns. Molecular species analysis showed a heterogeneous species composition for both human and bovine erythrocyte alkylacyl GroPIns. Their compositions are distinctly different from those of human and bovine erythrocyte acetylcholinesterase glycosyl-PtdIns anchors. The number of alkylacyl GroPIns molecules/cell is roughly equal with the number of glycosyl-PtdIns-anchored proteins in human erythrocytes.     

Determination of adenosine nucleotides in cultured cells by ion-pairing liquid chromatography-electrospray ionization mass spectrometry

A method using ion-pairing liquid chromatography-mass spectrometry (MS) was developed for analyzing adenosine 5(')-monophosphate (AMP), adenosine 5(')-diphosphate (ADP), and adenosine 5(')-triphosphate (ATP) in cellular extracts. Dimethylhexylamine (DMHA) was used as ion-pairing agent to retain and separate the analytes on a reversed-phase microbore column with a gradient program. Positive-ion electrospray ionization-MS was applied for the detection because of the use of the ion-pairing agent. Adduct ions of DMHA with AMP, ADP, and ATP were found to be the most intensive peaks and thus selected as quantitative ions. An external calibration method with linear ranges from 0.1 to 20 microM for AMP, 2 to 20 microM for ADP, and 2.5 to 20 microM for ATP was used for the quantitation. The method was applied to determine concentrations of AMP, ADP, and ATP in extracts of cultured rat C6 glioma cells that were pretreated with various concentrations of Zn. The detected levels of the adenosine nucleotides have been used to calculate total adenosine nucleotide and energy charge potential. Changes in cellular energy status upon exposure to increasing concentration of Zn in the culture medium were analyzed. The results indicated that the addition of Zn in a range of 40 to 120 microg/ml cause a gradual increased in energy charge potential of the cells.     

Quantitative determination of nitroglycerin in human plasma by capillary gas chromatography negative ion chemical ionization mass spectrometry

A quantitative method for determination of nitroglycerin in human plasma was developed. Nitroglycerin and the internal standard (butane-1,2,4-triyl trinitrate) were extracted from plasma with pentane. The extracts were analysed by gas chromatography mass spectrometry using fused silica capillary columns and electron capture negative ion chemical ionization. The quantitation limit of the method was about 50 pg ml-1. Linear calibration curves were obtained in the range of 50-1600 pg ml-1. Precision at the level of 100 pg ml-1 was 4%.     

Simultaneous quantification of arachidonic acid metabolites in cultured tumor cells using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry.

A validated method is described for the simultaneous analysis of PGE2, 11-, 12-, and 5-HETEs from cultured cells using HPLC negative electrospray ionization tandem mass spectrometry (LC/MS/MS). This method permits quantification of selected individual arachidonic acid metabolites from cell extracts without derivatization, multiple purification steps, or lengthy separation times required by traditional GC-MS- or HPLC-UV -based methods. Accuracy assessments of values calculated using this method showed deviations from nominal values were < or =15%. An average relative deviation of 7% of mean calculated values was observed for values taken on separate days. The lower limit of detection for all metabolites was 1.3 pg. The method was used to quantify arachidonic acid metabolites present in various cancer cell lines after incubation with arachidonic acid and the selective cyclooxygenase-2 inhibitor celecoxib. Results showed that the presence of celecoxib in lung cancer A549 cells reduced production of both PGE2 and 11-HETE in a concentration-dependent manner.     

Quantitative determination of tiamenidine hydrochloride in human serum using gas chromatography mass spectrometry

A method has been developed for the blood level determination of the antihypertensive agent tiamenidine hydrochloride. The serum samples are mixed with deuterium labelled tiamenidine hydrochloride as an internal standard and extracted with methylene chloride. The extracts are derivatized with heptafluorobutyric acid anhydride and analysed by means of gas chromatography mass spectrometry using the selected ion monitoring technique to measure the molecular ion intensities of the bis-heptafluorobutyryl derivatives of tiamenidine hydrochloride and of the internal standard. Using 5 ml serum, the limit of detection is 0.2 ng ml-1 with an accuracy of +/- 0.17 ng (Syx of the calibration curve).     

Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).

Ceramide (CER) is an important signaling molecule involved in a variety of cellular processes, including differentiation, cell growth, and apoptosis. Currently, different techniques are applied for CER quantitation, some of which are relatively insensitive and/or time consuming. Tandem mass spectrometry with its high selectivity and sensitivity is a very useful technique for detection of low abundant metabolites without prior purification or derivatization. In contrast to existing mass spectrometry methods, the developed electrospray tandem mass spectrometry (ESI-MS/MS) technique is capable of quantifying different CER species from crude cellular lipid extracts. The ESI-MS/MS is performed with a continuous flow injection and the use of an autosampler, resulting in a high throughput capability. The collision-induced fragmentation of CER produced, in addition to others, a characteristic fragment of m/z 264, making a precursor ion scan of 264 well suited for CER quantitation. Quantitation is achieved by use of a constant concentration of a non-naturally occurring internal standard C8-CER, together with a calibration curve established by spiking different concentrations of naturally occurring CER. The calibration curves showed linearity over a wide concentration range and sample volumes equivalent to 10 microg of cell protein corresponding to about 20, 000 fibroblasts were sufficient for CER analysis. Moreover this assay showed a detection limit at the subpicomole level. In summary, this methodology enables accurate and rapid analysis of CER from small samples without prior separation steps, thus providing a useful tool for signal transduction research.     

Validated semiquantitative/quantitative screening of 51 drugs in whole blood as silylated derivatives by gas chromatography-selected ion monitoring mass spectrometry and gas chromatography electron capture detection

A comprehensively validated procedure is presented for simultaneous semiquantitative/quantitative screening of 51 drugs of abuse or drugs potentially hazardous for traffic safety in serum, plasma or whole blood. Benzodiazepines (12), cannabinoids (3), opioids (8), cocaine, antidepressants (13), antipsychotics (5) and antiepileptics (2) as well as zolpidem, zaleplon, zopiclone, meprobamate, carisoprodol, tizanidine and orphenadrine and internal standard flurazepam, were isolated by high-yield liquid-liquid extraction (LLE). The dried extracts were derivatized by two-step silylation and analyzed by the combination of two different gas chromatographic (GC) separations with both electron capture detection (ECD) and mass spectrometry (MS) operating in a selected ion-monitoring (SIM) mode. Quantitative or semiquantitative results were obtained for each substance based on four-point calibration. In the validation tests, accuracy, reproducibility, linearity, limit of detection (LOD) and limit of quantitation (LOQ), selectivity, as well as extraction efficiency and stability of standard stock solutions were tested, and derivatization was optimized in detail. Intra- and inter-day precisions were within 2.5-21.8 and 6.0-22.5%, and square of correlation coefficients of linearity ranged from 0.9896 to 0.9999. The limit of quantitation (LOQ) varied from 2 to 2000 ng/ml due to a variety of the relevant concentrations of the analyzed substances in blood. The method is feasible for highly sensitive, reliable and possibly routinely performed clinical and forensic toxicological analyses.     

Determination of andrographolide in human plasma by high-performance liquid chromatography/mass spectrometry

In this paper, a rapid method based on high-performance liquid chromatography/electrospray-mass spectrometry (HPLC/ESI-MS) method for the quantitative determination of andrographolide (AND) in human plasma has been developed and validated. A liquid-liquid extraction (LLE) procedure was selected to isolate AND from biological matrixes. Isosorbide-5-mononitrate (IS-5-MN) was selected as the internal standard (IS). The correlation coefficient of the calibration curve was 0.998, in the range of 9.9-320.0 ng/mL. The validated method may be used to assess the bioavailability and pharmacokinetics of the drug.     

Preliminary application of liquid chromatography–electrospray-ionization mass spectrometry to the detection of 5-methyltetrahydrofolic acid monoglutamate in human plasma

Liquid chromatography (LC) in direct combination with mass spectrometry (MS) has been shown to be a good analytical technique for the selective separation and detection of labile folate monoglutamates. Reversed-phase LC and electrospray-ionization MS conditions were developed and optimized for the separation and detection of 5-methyltetrahydrofolic acid, 5-formyl tetrahydrofolic acid, tetrahydrofolic acid, dihydrofolic acid and folic acid in aqueous samples. Representative and reproducible positive ion mass spectra were generated for each folate under mild MS conditions. The selective MS detection and identification of endogenous 5-methyltetrahydrofolic acid in human plasma was accomplished through the development of a straightforward C₁₈-based solid-phase extraction procedure. This procedure allows for the qualitative assessment of 5-methyltetrahydrofolic acid in plasma. Based upon an isotope-dilution internal standard calibration study with standards, the LC–MS limit of quantitation for 5M-THF was estimated to be 0.39 ng/ml.     

Quantitative analysis of trimethylsilyl derivative of hydroxyurea in plasma by gas chromatography-mass spectrometry

Hydroxyurea is an antitumor drug widely used in the treatment of sickle cell disease. The drug has been analyzed in biological fluids by a number of high-performance liquid chromatography (HPLC) methods. This paper describes a fast and highly reliable capillary gas chromatography-mass spectrometry (GC-MS) procedure that was developed for the detection and quantitation of hydroxyurea in plasma. The compound and its labeled internal standard were liquid extracted from plasma and derivatized with BSTFA before analysis. The detection limit of the assay was 0.078 microg/ml and the limit of quantitation was 0.313 microg/ml with linearity up to 500 microg/ml. Intra-day variation, as coefficient of variation (C.V., %) over the selected concentration range, was 0.3-8.7% and inter-day variation was 0.4-9.6%.     

Quantitative determination of intact glucosinolates in broccoli, broccoli sprouts, Brussels sprouts, and cauliflower by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry

Many methods have been proposed to analyze glucosinolates, a class of phytochemicals whose breakdown products are thought to be responsible for an improvement in health; however, few are quantitative and many are time consuming. A selective and sensitive quantitative method for direct determination of intact glucosinolates was developed using liquid chromatography coupled to electrospray ionization tandem mass spectrometry with selected reaction monitoring detection. Detection limits for glucoiberin, sinigrin, progoitrin, glucoerucin, and glucotropaeolin were 1.75, 1.38, 1.36, 0.6, and 0.63 pmol, respectively. Intraassay precision of the method was within 10% for each compound. The method was successfully applied to quantify 10 individual glucosinolates in broccoli, broccoli sprouts, Brussels sprouts, and cauliflower. The advantage of the proposed method includes analysis of individual intact glucosinolates rather than the conversion to desulfoglucosinolates, an increased selectivity through the use of mass spectrometry, and a 10-fold improvement in detection sensitivity over conventionally used HPLC techniques.     

A comparison of thermospray and direct liquid introduction high-performance liquid chromatography/mass spectrometry for the analysis of candidate antimalarials

The analysis of antimalarials by high-performance liquid chromatography (HPLC)/mass spectrometry demonstrates a new dimension in specificity along with increased sensitivity compared to conventional HPLC detection methods. Both direct liquid introduction and thermospray HPLC/mass spectrometry interfaces provided molecular weight information as well as characteristic fragment ions for antimalarials not normally amenable to direct probe or gas chromatographic/mass spectrometric techniques. The direct liquid introduction interface, which incorporated a 1/100 split, showed a detection limit of 30 ng using selected ion monitoring. The thermospray technique showed less than 1 ng detection limits using selected ion monitoring.     

Quantification of steroid conjugates using fast atom bombardment mass spectrometry

Fast atom bombardment/mass spectrometry or liquid secondary ion mass spectrometry provides the capability for direct analysis of steroid conjugates (sulfates, glucuronides) without prior hydrolysis or derivatization. During the analysis of biologic extracts, limitations on the sensitivity of detection arise from the presence of co-extracted material which may suppress or obscure the analyte signal. A procedure is described for the quantitative determination of dehydroepiandrosterone sulfate in serum which achieved selective isolation of the analyte using immunoadsorption extraction and highly specific detection using tandem mass spectrometry. A stable isotope-labeled analog [( 2H2]dehydroepiandrosterone sulfate) was used as internal standard. Fast atom bombardment of dehydroepiandrosterone sulfate yielded abundant [M-H]- ions that fragmented following collisional activation to give HSO4-; m/z 97. During fast atom bombardment/tandem mass spectrometry of serum extracts, a scan of precursor ions fragmenting to give m/z 97 detected dehydroepiandrosterone sulfate and the [2H2]-labeled analog with a selectivity markedly superior to that observed using conventional mass spectrometry detection. Satisfactory agreement was observed between quantitative data obtained in this way and data obtained by gas chromatography/mass spectrometry of the heptafluorobutyrates of dehydroepiandrosterone sulfate and [2H2]dehydroepiandrosterone sulfate obtained by direct derivatization.     

Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification using tandem mass spectrometry

Modeling of metabolic networks as part of systems metabolic engineering requires reliable quantitative experimental data of intracellular concentrations. The hydrophilic interaction liquid chromatography–electrospray ionization–tandem mass spectrometry (HILIC–ESI–MS/MS) method was used for quantitative profiling of more than 50 hydrophilic key metabolites of cellular metabolism. Without prior derivatization, sugar phosphates, organic acids, nucleotides, and amino acids were measured under alkaline and acidic mobile phase conditions with pre-optimized multiple reaction monitoring (MRM) transitions. Irrespective of the polarity mode of the acquisition method used, alkaline conditions achieved the best quantification limits and linear dynamic ranges. Fully 90% of the analyzed metabolites presented detection limits better than 0.5 pmol (on column), and 70% presented 1.5-fold higher signal intensities under alkaline mobile phase conditions. The quality of the method was further demonstrated by absolute quantification of selected metabolites in intracellular extracts of Escherichia coli. In addition, quantification bias caused by matrix effects was investigated by comparison of calibration strategies: standard-based external calibration, isotope dilution, and standard addition with internal standards. Here, we recommend the use of alkaline mobile phase with polymer-based zwitterionic hydrophilic interaction chromatography (ZIC–pHILIC) as the most sensitive scenario for absolute quantification for a broad range of metabolites.     

Validation of an HPLC/MS/MS method with isotopic dilution for quantitative determination of trans,trans-muconic acid in urine samples of workers exposed to low benzene concentrations

Urinary trans,trans-muconic acid (t,t-MA), a biomarker of benzene exposure, is usually determined by HPLC methods with detection by either UV or, more recently, electrospray tandem mass spectrometry. However, not all these methods have been fully validated for quantitative analysis. This paper presents an HPLC/MS/MS method for reliable quantitative determination of t,t-MA that uses a commercial deuterium-labeled isotope as internal standard; the matrix effect has been evaluated and LOD is 0.22 microg/L. We used this method to test 200 urine samples, 175 of them collected at end-of-shift from workers in an oil refinery.     

Abscisic acid in dormant apple seed tissues - a rapid purification scheme using pre-packed columns and GCMS-SIM quantitation

A rapid small scale procedure has been developed for quantitative abscisic acid (ABA) determination by gas chromatography - mass spectrometry with selected ion monitoping (GCMS-SIM). Extracts of apple seeds ( Malus domestica cv. Northern Spy) were passed first through 3 ml C₁₈ columns, and then through 3 ml silica gel columns. GCMS-SIM quantitation of ABA was done by adding an internal standard, hexadeuterated ABA. Ten sample extracts could be purified and analyzed by GCMS in 5 to 6 h, offering a quick and precise method to laboratories equipped with GC-MS instrumentation. The endosperm membrane and seed coat of apple seeds contained 2.5-3 times the ABA concentration found in the embryonic axis and cotyledons, supporting the hypothesis that it may be the ABA content of the membrane and seed coat that explains their inhibitory qualities with respect to seed germination.     

An Improved UPLC-MS/MS Platform for Quantitative Analysis of Glycerophosphoinositol in Mammalian Cells

The glycerophosphoinositols constitute a class of biologically active lipid-derived mediators whose intracellular levels are modulated during physiological and pathological cell processes. Comprehensive assessment of the role of these compounds expands beyond the cellular biology of lipids and includes rapid and unambiguous measurement in cells and tissues. Here we describe a sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative analysis of the most abundant among these phosphoinositide derivatives in mammalian cells, the glycerophosphoinositol (GroPIns). The method has been developed in mouse Raw 264.7 macrophages with limits of quantitation at 3 ng/ml. Validation on the same cell line showed excellent response in terms of linear dynamic range (from 3 to 3,000 ng/ml), intra-day and inter-day precision (coefficient of variation ≤ 7.10%) and accuracy (between 98.1 and 109.0%) in the range 10-320 ng/ml. As proof of concept, a simplified analytical platform based on this method and external calibration was also tested on four stimulated and unstimulated cell lines, including Raw 264.7 macrophages, Jurkat T-cells, A375MM melanoma cells and rat basophilic leukemia RBL-2H3 cells. The results indicate a wide variation in GroPIns levels among different cell lines and stimulation conditions, although the measurements were always in line with the literature. No significant matrix effects were observed thus indicating that the here proposed method can be of general use for similar determinations in cells of different origin.     

Strategy of glycerolipid separation and quantitation by complementary analytical techniques : Plenary lecture

Although improved systems for chromatographic resolution continue to be developed there is good reason to believe that no single method will be capable of complete separation of all lipid mixtures including the geometric, positional and stereochemical isomers in each molecular species. Furthermore, the chromatographic systems giving the highest resolution usually yield the least complete recoveries of components and require separate procedures of quantitation. It is therefore necessary to develop appropriate strategies that yield the required resolution as a result of consecutive application of complementary analytical techniques. At the present time, the original combination of thin-layer and gas— liquid chromatography has been joined by the combination of thin-layer and liquid, and liquid and gas—liquid chromatography with both liquid and gas—liquid chromatography being frequently coupled to mass spectrometry with computerized data processing. Internal standardization with hydrogen flame ionization provides a simple quantitative detection for gas chromatography, while mass spectrometry serves a similar purpose in liquid chromatography, although a much more extensive calibration may be required for quantitation. Special advantages for both separation and quantitation of most neutral lipid mixtures are derived from enzymic and chemical modification of the samples prior to chromatography. With imaginative work-up of samples, superior qualitative and quantitative results can frequently be obtained by appropriate combination of chromatographic techniques of limited resolving power.     

Quantitative analysis of albuterol in human plasma by combined gas chromatography chemical ionization mass spectrometry

A highly sensitive and specific quantitative assay for the determination of albuterol in human plasma, based on selected ion monitoring gas chromatography chemical ionization mass spectrometry, has been developed. The [MH]+ ions from the tri-TMS derivatives of albuterol (m/z 456) and the internal standard (2H3)albuterol (m/z 459), were assayed simultaneously by selected ion monitoring. The lower limit of quantitation is 0.25 ng ml-1 and the average assay precision (CV) for albuterol concentrations ranging from 0.25 ng ml-1 to 25 ng ml-1 is approximately 4%. This method is currently being employed for the routine quantitation of albuterol in plasma following the administration of doses therapeutically effective to man.     

Selective determination of sultopride in human plasma using high-performance liquid chromatography with ultraviolet detection and particle beam mass spectrometry

We developed a sensitive and selective method for determining levels of sultopride, a neuroleptic drug of the substituted benzamide, in human plasma using high-performance liquid chromatography (HPLC) combined with UV detection and particle beam mass spectrometry (PBMS). Sutopride was extracted with tert.-butylmethyl ether using a salting-out technique. Tiapride served as an internal standard (I.S.). Sutopride and I.S. were separated by HPLC on a silica column with a mobile phase of acetonitrile-0.1 M ammonium acetate (94:6, v/v). The calibration curves were linear over the concentration range from 5 to 1000 ng/ml by HPLC with UV detection and from 10 to 1000 ng/ml with PBMS detection. The limit of quantitation was 5 ng/ml with UV detection and 10 ng/ml with PBMS detection. The absolute recovery was 92% and the within-day coefficients of variation were 2.9–7.1% at plasma concentrations from 50 to 500 ng/ml, determined by HPLC with UV detection. Using this method, we measured the plasma concentrations of sultopride with replicate analyses in four hospitalized patients and steady-state plasma levels were determined to be 161.6±30.8, 321.1±93.7, 726.5±143.1 and 1273.6±211.2 ng/ml, respectively.