Simultaneous quantitative analysis of methyl salicylate,ethyl salicylate and salicylic acid from biological fluids using gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric (GC–MS) assay was developed for the quantitative analysis of methyl salicylate (MeS), ethyl salicylate (ES) and salicylic acid (SA) from biological fluids. The method was validated from 100-μl rat liver homogenate preparations (5 mg/ml protein) in 70 mM KH₂PO₄ (pH 7.4) buffer and from 100 μl rat plasma. The samples were extracted with chloroform, derivatized with BSTFA and quantitated by GC–MS in the SIM mode. The standard curves ranged from 31 ng/ml to 800 or 1250 ng/ml. Relative standard deviations and bias were less than 11% in plasma and homogenate for all compounds except SA which evidenced greater variability. The assay was used in preliminary experiments to characterize the pharmacokinetics of MeS in rats.     

An automated fluorescence assay for subnanogram quantities of protein in the presence of interfering material

An automated assay for measuring nanogram and subnanogram quantities of protein in microliter samples was developed with the fluorometric reagent omicron-phthaldialdehyde/mercaptoethanol. Low molecular weight interfering substances were separated within the analysis by gel filtration. The technique allowed measurement of biological fluids without any sample pretreatment. The method presented proved to be linear within the range 1.2 ng to 1.4 microgram with a standard deviation of +/- 4.2%. The minimum detectable protein concentration was 1 microgram/liter. Special care was taken to prevent any determination errors caused by losses of protein during adsorption to surfaces. The simple analytical apparatus constructed can be used for field studies and ran several weeks when continuously used for seawater analysis aboard ship.     

Quantification of isoflavones and lignans in urine using gas chromatography/mass spectrometry

Phytoestrogens (isoflavones and lignans) are of increasing interest due to their potential to prevent certain types of complex diseases. However, epidemiological evidence is needed on the levels of phytoestrogens and their metabolites in foods and biological fluids in relation to risk of these diseases. We report an assay for phytoestrogens which is sensitive, accurate, and uses low volumes of sample. Suitable for epidemiological studies, the assay consists of a simple sample preparation procedure and has been developed for the analysis of five isoflavones (daidzein, O-desmethylangolensin, equol, genistein, and glycitein) and two lignans (enterodiol and enterolactone), which requires only 200 microl of urine and utilizes one solid-phase extraction stage for sample preparation prior to derivatization for GC/MS analysis. Limits of detection were in the region 1.2 ng/ml (enterodiol) to 5.3ng/ml (enterolactone) and the method performed well in the UK Government's Food Standards Agency-sponsored quality assurance scheme for phytoestrogens. For the first time, average levels of all the above phytoestrogens were measured in samples of urine collected from a free living population sample of women. Results show a large range in both the amount and the type of phytoestrogens excreted.     

Quantitative measurement of a new synthetic hetrazepine derivative, BN50730, in human plasma and urine by combined liquid chromatography—negative chemical ionization mass spectrometry using a particle beam interface

A new simple and sensitive assay has been developed for the quantitative measurement of BN50730 at the picomole level in human plasma and urine. The drug and the internal standard (BN50765) were measured by combined liquid chromatography—negative chemical ionization mass spectrometry with methane as the reagent gas. A simple solid—liquid extraction procedure was used to isolate BN50730 from complex biological matrices. Mild operating conditions were required to assay the parent drug with a particle beam interface from Hewlett-Packard. The mass spectrometer was tuned to monitor the intense ion m/z 333, which was generated in the ion source by a dissociative capture process. This assay was performed with 1 ml of plasma or 0.1 ml of urine, and the quantification limit of the method was statistically calculated as 1 ng ml⁻¹. The very low relative standard deviation and mean percentage of error calculated during the different within-day or between-day repeatability assays clearly demonstrate the ruggedness of the technique for the routine determination of BN50730 in the biological fluids. Some preliminary results on the pharmacokinetics of the drug are presented to illustrate the applicability of this new powerful LC—MS method.     

Artifact-Free Quantification of Free 3-Chlorotyrosine, 3-Bromotyrosine, and 3-Nitrotyrosine in Human Plasma by Electron Capture–Negative Chemical Ionization Gas Chromatography Mass Spectrometry and Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry

Halogenation and nitration of biomolecules have been proposed as key mechanisms of host defense against bacteria, fungi, and viruses. Reactive oxidants also have the potential to damage host tissue, and they have been implicated in disease. In the current studies, we describe specific, sensitive, and quantitative methods for detecting three stable markers of oxidative damage: 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine. Our results indicate that electron capture-negative chemical ionization-gas chromatography/mass spectrometry (EC-NCI GC/MS) is 100-fold more sensitive than liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) for analyzing authentic 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine. Using an isotopomer of tyrosine to evaluate artifactual production of the analytes during sample preparation and analysis, we found that artifact generation was negligible with either technique. However, LC-MS/MS proved cumbersome for analyzing multiple samples because it required 1.5 h of run and equilibration time per analysis. In contrast, EC-NCI GC/MS required only 5 min of run time per analysis. Using EC-NCI GC/MS, we were able to detect and quantify attomole levels of free 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine in human plasma. Our results indicate that EC-NCI GC/MS is a sensitive and specific method for quantifying free 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine in biological fluids in a single, rapid analysis and that it avoids generating any of the analytes ex vivo.     

Determination of three phthalate metabolites in human urine using on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry

An on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry (on-line SPE-HPLC-MS/MS) method was developed for the analysis of metabolites of three phthalate esters in human urine at the low nanogram per milliliter level. The recoveries were above 84.3% and relative standard deviations varied from 0.8 to 4.8%. The compounds along with their deuterated internal standards were detected in the negative ion mode by selective reaction monitoring and the accuracy of the method was improved by isotope dilution. Monobutyl phthalate was detected with median level of 22.5 ng/ml. The median levels for monobenzyl phthalate and monoethylhexyl phthalate were less than the limit of quantitation (LOQ). The on-line SPE-HPLC-MS/MS method allowed the possibility of determining these metabolites within a short time, with increased sensitivity and by using decreased amounts of sample and solvent.     

Single-step procedure for gas chromatography-mass spectrometry screening and quantitative determination of amphetamine-type stimulants and related drugs in blood, serum, oral fluid and urine samples

We describe a rapid GC/MS assay for amphetamine-type stimulant drugs (ATSs) and structurally related common medicaments in blood, serum, oral fluid and urine samples. The drugs were extracted from their matrices and derivatized with heptafluorobutyric anhydride (HFBA) in a single step, using the following procedure: 100 microl (oral fluid) or 200 microl (blood, serum, urine) of the sample were mixed with 50 microl of alkaline buffer and 500 microl of extraction-derivatization reagent (toluene + HFBA + internal standard), centrifuged, and injected into a GC/MS apparatus. As revealed by the validation data this procedure, with its limit of quantitation being set at 20 ng/ml for oral fluid, 25 ng/ml for blood or 200 ng/ml for urine, is suitable for screening, identification and quantitative determination of the ATSs and related drugs in all the matrices examined. Thus, time-consuming and expensive multiple analyses are not needed, unless specifically required.     

Quantification of 20-hydroxyeicosatetraenoic acid by colorimetric competitive enzyme linked immunosorbent assay

Analysis of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor produced by the cytochrome P450 pathway, presently requires high-performance liquid chromatography (HPLC) and gas chromatography/ mass spectrometry (GC/MS). To simplify 20-HETE analysis, competitive ELISAs were developed using polyclonal anti-20-HETE coated ELISA plates to which free 20-HETE and 20-HETE conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) were added. Assays were developed with and without a pro prietary enhancer solution which allows for the extraction-free measurement of 20-HETE in urine samples. The bound 20-HETE-HRP or 20-HETE-AP was detected using 3,3 ,5,5, -tetramethylbenzidine and p-nitrophenyl phosphate, respectively. Sensitivities expressed as 80% B/B0, were 0.1 ng/ml for the HRP assay, and 0 5 ng/ml for the AP assay, with r2 = 0 99 for both formats. Of the 17 lipids tested for cross-reactivity, arachidonic acid showed the highest (0.32%) followed by racemic 5-HETE (0.07%) and 8,9-dihydroxyeicosatrienoic acid (DHET) (0.04%). Preliminary validation experiments examining serum and urine concentrations of 20-HETE yield values that fall within the ranges established by GC/MS in the literature. These ELISAs provide simple and inexpensive methods for the analysis of 20-HETE in biological samples.     

Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution

Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1-10 ng/ml range with percent coefficients of variation from 3 to 15% without immunoaffinity enrichment of either proteins or peptides. Sample processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate biomarker proteins were developed and optimized by addition of exogenous proteins to immunoaffinity depleted plasma from a healthy donor. Quantitative multiple reaction monitoring assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concentrations of spiked protein in plasma, we determined that each target protein had at least one signature peptide with a limit of quantitation in the 1-10 ng/ml range and linearity typically over 2 orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/milliliter range. These levels of assay performance represent up to a 1000-fold improvement compared with direct analysis of proteins in plasma by MS and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatography at the peptide level prior to LC-multiple reaction monitoring/MS. The methods presented here provide a solid basis for developing quantitative MS-based assays of low level proteins in blood.     

Quantification of dinor, dihydro metabolites of F2-isoprostanes in urine by liquid chromatography/tandem mass spectrometry

The F2-isoprostanes (F2-IsoP) are a series of prostaglandin (PG)-F2-like compounds that are produced by free-radical-mediated oxidation of arachidonic acid. One F2-IsoP with potent biological activity is 15-F2t-IsoP and increased levels of 15-F(2t)-IsoP have been measured in several diseases. The major urinary metabolite of 15-F2t-IsoP (8-iso-PGF(2alpha)) is 2,3-dinor-5,6-dihydro-15-F2t-IsoP (15-F2t-IsoP-M). Previously, we developed a stable isotope dilution gas chromatography/negative chemical ionization/mass spectrometry (MS) assay for 15-F2t-IsoP-M, which, while highly sensitive, required time-consuming derivatization and thin-layer chromatography purification. We now report the development of a more rapid high-performance liquid chromatography method coupled to electrospray ionization-tandem mass spectrometry (LC/MS/MS) to analyze all of the dinor,dihydro metabolites of the F2-IsoP isomers (F2-IsoP-M). The precision of this assay was +/-5.0% and the accuracy 80%. The assay remained linear over a range of 1-100 ng injected onto the LC column. Levels of F2-IsoP-M determined by the LC/MS/MS assay method significantly correlated with levels of 15-F2t-IsoP-M determined by the GC/MS assay (R = 0.77y = 67.2x-0.5). The levels of F2-IsoP-M detected in spot urines from 40 normal subjects were 38.1+/-19.1 ng/mg creatinine (mean+/-SD). This method provides an accurate and rapid assay to assess oxidative status in vivo.     

Chromatographic and immunochemical approaches to the analysis of the HIV protease inhibitor saquinavir in plasma

The development of the HIV protease inhibitor saquinavir (Ro 31-8959) required a range of analytical methods for its measurement in biological fluids. This paper describes the development of isocratic, reverse-phase HPLC/UV methods for the routine measurement of plasma levels of the drug together with a more sensitive radioimmunoassay. The performance of the two assays is compared with that of an HPLC/MS/MS method previously published and has been shown to be satisfactory, with coefficients of variation of calibration standards and quality control samples within the usual outside limits of +/- 15%. The HPLC/UV method can be routinely applied for concentrations down to 10-20 ng/ml and a lower limit of quantification of 1 ng/ml from 1 ml of human plasma is possible. The radioimmunoassay was developed for the specific measurement of saquinavir concentrations in human, HIV-positive plasma samples and has a lower limit of quantification of 0.5-1.0 ng/ml. Some preliminary findings suggested that it might not be specific in rat plasma and no attempts have been made to quantify any nonclinical samples with this technique. If still greater sensitivity is required, recourse can be made to the HPLC/MS/MS assay.     

Determination of pentacaine, trans-2-(1-pyrrolidinyl)cyclohexyl-3-pentyloxycarbanilate hydrochloride, in biological samples by gas chromatography/mass spectrometry

A quantitative and selective method has been developed for the determination of a novel local anaesthetic compound pentacaine, trans-2-(1-pyrrolidinyl)cyclohexyl-3-pentyloxycarbanilate hydrochloride, in biological samples. After ion pair extraction from 1 M HCl into 1,2-dichloroethane, pentacaine and a structurally related internal standard were derivatized to prevent thermal decomposition in the gas chromatograph. An on-column methylation technique with trimethylanilinium hydroxide was used. Determination was performed by gas chromatography/mass spectrometry (GC/MS) with selected ion monitoring. Interferences by endogenous lipophilic constituents were avoided by including an n-hexane wash before the ion pair extraction. This wash step did not reduce the drug recoveries. The method gave linear results over a concentration range of 5-100 ng ml-1 with a coefficient of variation less than 10% at 5 ng pentacaine ml-1. Specimens of plasma, whole blood, urine as well as in vitro preparations such as hepatic microsomes were successfully analysed.     

Quantitation of vitamin B6 in biological samples by isotope dilution mass spectrometry

Methods have been developed for the simultaneous quantitative analysis of vitamin B6 forms in biological samples by isotope dilution mass spectrometry using deuterated forms of pyridoxine, pyridoxal, pyridoxamine, and pyridoxic acid. The biological fluid or tissue sample was homogenized and then treated with a cocktail containing appropriate amounts of each deuterated vitamer, as well as the deuterated, phosphorylated vitamer forms. The individual vitamers were isolated from the homogenate by a complex high-performance liquid chromatographic procedure that provided separate fractions for each of the six vitamers found in biological samples. Aldehydic B6 vitamers were reduced to the alcohol form prior to acetylation and analysis by gas chromatography/mass spectrometry (GC/MS). The three resulting vitamers were analyzed by electron ionization GC/MS using a silicone capillary column. The methods have been applied to analysis of vitamin B6 in liver, milk, urine, and feces at levels as low as 0.02 nmol/ml.     

A novel method for the determination of 1,5-anhydroglucitol, a glycemic marker, in human urine utilizing hydrophilic interaction liquid chromatography/MS(3)

Plasma levels of 1,5-anhydroglucitol (1-deoxyglucose), a short-term marker of glycemic control, have been measured and used clinically in Japan since the early 1990s. Plasma levels of 1,5-anhydroglucitol are typically measured using either a commercially available enzymatic kit or GC/MS. A more sensitive method is needed for the analysis of 1,5-anhydroglucitol in urine, where levels are significantly lower than in plasma. We have developed a sensitive and selective LC/MS(3) assay utilizing hydrophilic interaction liquid chromatography and ion trap mass spectrometry for the quantitative determination of 1,5-anhydroglucitol in human urine. Diluted human urine samples were analyzed by LC/MS(3) using an APCI source operated in the negative ionization mode. Use of an ion trap allowed monitoring of MS(3) transitions for both 1,5-anhydroglucitol and the internal standard which provided sufficient selectivity and sensitivity for analysis from 50 microL of human urine. Quantitation of 1,5-anhydroglucitol levels in urine was accomplished using a calibration curve generated in water (calibration range 50 ng/mL to 10 microg/mL). Method ruggedness and reproducibility were evaluated by determining the intra- and inter-day accuracies and precision of the assay, as well as the bench-top and freeze-thaw stability. For both inter- and intra-assay evaluations, the accuracy of the assay was found to be acceptable, with the concentrations of all QCs tested not deviating more than 8% from theoretical. Four-hour bench-top and freeze-thaw stabilities were also evaluated; 1,5-anhydroglucitol was found to be stable at room temperature (<18% deviation from theoretical) and during 3 freeze-thaw cycles (<1% deviation from theoretical, except at the lowest QC level). The LC/MS(3) assay was then used to successfully determine the concentration of 1,5-AG in more than 200 urine samples from diabetic patients enrolled in a clinical study.     

LC-MS/MS detection of peroxynitrite-derived 3-nitrotyrosine in rat microvessels

3-Nitrotyrosine (3NT) is used as a biomarker of nitrative pathology caused by peroxynitrite (PN), myeloperoxidase (MPO)-, and/or eosinophil peroxidase (EPO)-dependent nitrite oxidation. 3NT measurements in biological materials are usually based on either antibody staining, HPLC detection, or GC detection methodologies. In this report, a procedure is described for the measurement of 3NT and tyrosine (TYR) by LC-MS/MS that is simple, direct, and sensitive. Though highly specialized in its use as an assay, LC-MS/MS technology is available in many research centers in academia and industry. The critical assay for 3NT was linear below 100 ng/ml and the limit of detection was below 100 pg/ml. Regarding protein digested samples, we found that MRM was most selective with 133.1 m/z as the daughter ion. In comparison, LC-ECD was 100 times less sensitive. Basal levels of 3NT in extracted digests of rat brain homogenate were easily detected by LC-MS/MS, but were below detection by LC-ECD. The LC-MS/MS assay was used to detect 3NT in rat brain homogenate that was filtered through a 180 micron nylon mesh. Three fractions were collected and examined by phase contrast microscopy. The mass ratio (3NT/TYR) of 3NT in fractions of large vessel enrichment, microvessel enrichment, and vessel depletion was 0.6 ng/mg, 1.2 ng/mg, and 0.2 ng/mg, respectively. Ultimately, we found that the basal 3NT/TYR mass ratio as determined by LC-MS/MS was six times greater in microvessel-enriched brain tissue vs. tissue devoid of microvessels.     

双抗体夹心法检测3-硝基酪氨酸方法的建立及应用

目的：建立一种准确、快速的双抗体夹心酶免疫吸附的方法,以定量检测组织中过氧亚硝基阴离子的水平。方法：分别以小鼠源性抗3-硝基酪氨酸单克隆IgG抗体和兔源性抗3-硝基酪氨酸IgG抗体为包被抗体和检测抗体,采用正交设计方法摸索以上各抗体的浓度,建立定量检测3-硝基酪氨酸（3-NT）的双抗体夹心ELISA法。同时,测定心肌缺血再灌注大鼠心肌组织3-NT的含量。结果：本研究建立的双抗体夹心ELISA法检测3-NT的最低检测下限为0．10ng·ml^-1,具有良好线性关系的检测范围是（0．15-7．50）ng·ml^-1（r^2=0．995）;心肌缺血再灌注组大鼠的心肌组织中的3-NT水平为（1022．42±97．35）ng·mg pro^-1,明显高于假手术组（246．58±56．52ng·mg pro^-1,P〈0．01）。结论：本研究建立的定量检测3-NT的双抗体夹心ELISA法能够方便、准确、快速地检测组织中3-NT的含量,为定量检测组织中ONOO-的水平提供了新的方法。     

Sensitive determination of pethidine in body fluids by surface ionization organic mass spectrometry

We have presented a simple and sensitive method for determining pethidine, a narcotic analgesic drug in body fluids by gas chromatography (GC)/surface ionization organic mass spectrometry (SIOMS). Good linearity was obtained in the range of 0.625–25 ng/ml of whole blood and urine by mass chromatography, and in the range of 0.05–2 ng/ml of whole blood by selected ion monitoring (SIM). Pethidine and diphenylpyraline (internal standard) were extracted from body fluids with Bond Elut Certify cartridges; their recoveries were above 95%. The detection limits (signal-to-noise ratio=3) were estimated to be 0.2 ng/ml of whole blood or urine by mass chromatography, 0.02 ng/ml of whole blood by SIM.     

Quantification of endocannabinoids in rat biological samples by GC/MS: technical and theoretical considerations

In the last several years, interest has increased significantly about the endocannabinoids anandamide and 2-arachidonylglycerol, two lipid messengers that activate cannabinoid receptors. Quantification of these compounds in biological samples presents numerous technical challenges. Because of their low abundance, endocannabinoids are usually quantified by isotope dilution assays using mass spectrometry coupled to either gas chromatography or high-performance liquid chromatography. Although endocannabinoid levels in biological fluids, such as plasma and cerebrospinal fluid, can be directly determined by these techniques, the complex lipid profile of brain tissue samples mandates purification of lipid extracts before GC/MS analysis; this step is not necessary when using HPLC/MS. We have found that when silica gel chromatography is used for endocannabinoid purification, poor recovery and loss of deuterium from the internal standards lead to inaccurate estimation of endocannabinoid levels. By contrast, purification strategies using C(18) solid-phase extraction permits precise and reproducible GC/MS quantification of endocannabinoids in tissue samples.     

Determination of chlorpromazine and its major metabolites by gas chromatography/mass spectrometry: application to biological fluids

A method for the quantitative determination of chlorpromazine and five of its major metabolites in a single sample of biological fluid in the ng/ml range has been developed utilizing gas chromatography/mass spectrometry with selected ion recording. The assay is highly specific and quantification is accomplished by an inverse stable isotope dilution technique, using deuterium-labeled variants of the compounds as internal standards. In this way the concentrations of chlorpromazine and five of its major metabolites (the sulfoxide, the N-oxide, the monodemethylated, the didemethylated, and the 7-hydroxylated compounds) can be determined in biological fluids. Levels in humans have been measured both in plasma and in red blood cells and are compared to those found in related in vitro studies.     

Immunoaffinity liquid chromatography-tandem mass spectrometry detection of nitrotyrosine in biological fluids: development of a clinically translatable biomarker

Measurement of nitrotyrosine levels in biological fluids can serve as a biomarker for oxidative/nitrative damage arising from formation of reactive nitrogen species, including peroxynitrite. Peroxynitrite is formed by the reaction of the superoxide radical (O2.-) with the nitric oxide radical (.NO) that is generated by nitric oxide synthase (NOS). This article describes an immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure 3-nitrotyrosine at very low (picomolar) levels. Incorporation of a pronase digestion step prior to the immunoaffinity LC-MS/MS allowed for measuring not only free amino acid but also protein 3-nitrotyrosine in biological fluids. The use of an in-line antibody column allowed for increased specificity as compared with previously reported assays. The assay is linear over a range of 5 to 500 pg/ml (0.022-2.20 nM, r(2)=0.9987), with the lower detection limit being 5 pg/ml. In addition to its increased sensitivity and specificity, this assay showed great nitrotyrosine recovery from biological fluids when either nitrotyrosine or nitrotyrosine-containing peptides were added exogenously. The utility of this assay for nitrotyrosine as a clinically translatable biomarker was demonstrated by quantifying both free and total nitrotyrosine levels in various biological fluids, including urine, plasma, serum, cerebrospinal fluid (CSF), and synovial fluid (SF) from both preclinical species and human subjects. Thus, whether in an animal model of human disease or in a clinical setting, the quantification of nitrotyrosine levels should provide support for NOS-driven pathology and its blockade following therapeutic intervention.