Journal of Chromatography B (v.877, #25)

33rd Annual Meeting of the Japanese Society for Biomedical Mass Spectrometry by Ryo Taguchi; Toshimitsu Niwa; Tomiko Kuhara (2599).

Mass spectrometry (MS) has been successfully applied for the identification and quantification of uremic toxins and uremia-associated modified proteins. This review focuses on recent progress in the analysis of uremic toxins by using MS. Uremic toxins include low-molecular-weight compounds (e.g., indoxyl sulfate, p-cresol sulfate, 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid, asymmetric dimethylarginine), middle-molecular-weight peptides, and proteins modified with advanced glycation and oxidation. These uremic toxins are considered to be involved in a variety of symptoms which may appear in patients with stage 5 chronic kidney disease. Based on MS analysis of these uremic toxins, the pathogenesis of the uremic symptoms will be elucidated to prevent and manage the symptoms.
Keywords: Mass spectrometry; Uremic toxin; Modified proteins; Chronic kidney disease; Uremia;

For the purpose of biomarker discovery, we originally developed a novel method for quantitative proteome analysis utilizing both tryptophan-targeted stable isotope tagging and mass spectrometry. The method has now been refined by replacing detergents and an enrichment column and further utilizing a novel matrix that is specifically suitable for tagged peptides. A total analytical system has been constructed by combining this method with HPLC, an automatic spotter, MALDI-TOF MS and analytical software. Clinical tissue samples such as colorectal carcinoma and renal cell carcinoma were analyzed using this system, and the results demonstrated that it is useful for discovering novel biomarker candidates. Here, we review a series of these studies and also discuss future directions for development of this technology.
Keywords: Quantitative proteome; Biomarker discovery; Stable isotope; NBS method;

Simultaneous determination of salivary testosterone and dehydroepiandrosterone using LC–MS/MS: Method development and evaluation of applicability for diagnosis and medication for late-onset hypogonadism by Yujin Shibayama; Tatsuya Higashi; Kazutake Shimada; Akira Odani; Atsushi Mizokami; Hiroyuki Konaka; Eitetsu Koh; Mikio Namiki (2615-2623).
Late-onset hypogonadism (LOH) is a male-specific disorder caused by the age-related decline in androgens, such as testosterone (T). A sensitive liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS/MS) method for the simultaneous quantification of T and its precursor, dehydroepiandrosterone (DHEA), in human saliva has been developed and validated. The saliva was deprotenized with acetonitrile, purified using a Strata-X cartridge, derivatized with 2-hydrazino-1-methylpyridine, and subjected to LC–MS/MS. The recovery rates of the steroids during the pretreatment were about 90%. Quantification was based on selected reaction monitoring using characteristic transitions, and deuterated T and DHEA were used as internal standards. This method allowed the reproducible (inter- and intra-assay precisions, <2.9%) and accurate (accuracy, 98.5–101.8%) quantification of the salivary androgens using a 500-μl sample and the limits of quantification for both androgens were 10 pg/ml. As preliminary steps in the practical application of the developed method in diagnosis and medication for LOH, the diurnal rhythms, inter-day alternations and age differences in the salivary T and DHEA were examined; the method found that the salivary T and DHEA show specific diurnal rhythms, significant alternations in early morning and pronouncedly decline with age. The method also enabled the determination of the changes in the individual T and DHEA levels after the DHEA supplementation, which is expected to be a new and easy medication for LOH. Thus, the developed method has satisfactory applicability in the diagnosis and medication for LOH.
Keywords: Testosterone; Dehydroepiandrosterone; Saliva; Late-onset hypogonadism; Liquid chromatography–electrospray ionization-tandem mass spectrometry; Simultaneous quantification;

Inorganic arsenic species are metabolized to monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV) and excreted into urine. A simple, rapid and sensitive method has been developed using electrospray ionization tandem mass spectrometry (ESI-MS–MS) for the simultaneous determination of MMAV and DMAV. MMAV and DMAV in a sample were allowed to react with citric acid (CiA). Adduct compounds were extracted together with isoamyl alcohol (IAA). An aliquot (1-μL) of the IAA layer was directly injected into the ESI-MS–MS instrument, and was detected within 1 min. Quantification was done using selected reaction monitoring for MMAV and DMAV as follows: [MMAH + 2CiA − 3H2O]+  → [MMAH + CiA − 2H2O]+ [DMAH + CiA + MeOH − 2H2O]+  → [DMAH + MeOH − H2O]+ where MMAH and DMAH denote the protonated forms of MMAV and DMAV, and MeOH denotes methanol (carrier liquid in ESI-MS–MS). This method was validated for the analysis of urine samples. The limit of detection of As was 0.3 μg L−1 for MMAV and 0.6 μg L−1 for DMAV using 10 μL of sample solution. Results were obtained in <10 min with a linear calibration range of 3–100 μg L−1. Inorganic arsenic compounds (and other organic arsenic compounds) found in urine did not interfere with the detection of MMAV and DMAV. Concentrations of MMAV and DMAV in the reference urine SRM 2670a were estimated after partial purification, and those in urine of a patient treated with As2O3 were measured after dilution.
Keywords: Monomethylarsonic acid; Dimethylarsinic acid; Tandem mass spectrometry; Electrospray ionization; Citric acid; Adduct;

Chemical synthesis of N-acetylcysteine conjugates of bile acids and in vivo formation in cholestatic rats as shown by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry by Kuniko Mitamura; Saai Watanabe; Toshihiro Sakai; Rika Okihara; Mitsuru Sogabe; Tateaki Wakamiya; Alan F. Hofmann; Shigeo Ikegawa (2630-2638).
N-Acetylcysteine (NAC) conjugates of the five major bile acids occurring in man were synthesized in order to investigate the possible formation in vivo of these conjugates. Upon collision-induced dissociation, structurally informative daughter ions were observed. The transformation of cholyl-adenylate and cholyl-CoA thioester into a N-acetyl-S-(cholyl)cysteine by rat hepatic glutathione S-transferase was confirmed by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry (LC/ESI-MS2). Lithocholic acid was administered orally to bile duct-ligated rats that also received NAC intraperitoneally. The NAC conjugate of lithocholic acid was identified in urine by means of LC/ESI-MS2. Rapid hydrolysis of the BA-NAC conjugates by rabbit liver carboxylesterase was found, demonstrating the possible labile nature of the NAC conjugates formed in the liver.
Keywords: Bile acid; N-Acetylcysteine; Glutathione S-transferase; Rat urine; Carboxylesterase; Liquid chromatography/electrospray ionization-mass spectrometry;

Recently, global analysis of triacylglycerols (TAGs) has become increasingly important in studies of abnormality of lipid metabolism in metabolic syndrome. TAGs consist of various molecular species, caused by their three fatty acyl chains with a large variety of carbon chain lengths and degrees of unsaturation. Therefore, most previously reported methods have been insufficient in global detection of TAGs including their structural isomers and TAGs with oxidized or odd number acyl carbon chain. Here we report an effective method for global analysis of TAG molecular species from complex lipid mixtures of mouse liver and white adipose tissue (WAT) using reverse-phased high resolution liquid chromatography (LC) coupled with electrospray ionization (ESI)-quadrapole/time of flight hybrid mass spectrometer (QTOF-MS). For effective profiling of TAG molecular species, sensitive two-dimensional (2D) maps were constructed and individual structures were correctly identified by the elution profile and MS/MS. As a result, TAGs including their structural isomers and TAGs with an odd number acyl carbon chain were separated and detected effectively on the 2D map as compared with conventional high performance LC. It was also found that our 2D profiling method was useful in searching characteristic molecular species globally. In mouse WAT, novel oxidized TAGs, which were mainly formed by hydroperoxidation of one of their linoleic acyl chains, were effectively detected in comparison with TAG molecular species of mouse liver.
Keywords: Triacylglycerol; Global analysis; Two-dimensional profiling; Lipidomics;

Enzymatic evaluation of glutaric acidemia type 1 by an in vitro probe assay of acylcarnitine profiling using fibroblasts and electrospray ionization/tandem mass spectrometry (MS/MS) by Yuichi Mushimoto; Yuki Hasegawa; Hironori Kobayashi; Hong Li; Jamiyan Purevsuren; Isamu Nakamura; Takeshi Taketani; Seiji Fukuda; Seiji Yamaguchi (2648-2651).
Glutaric acidemia type 1 (GA1) is usually diagnosed with an accumulation of glutaric acid (GA) or 3-hydroxyglutaric acid by GC/MS. In some cases, however, excretion of GA is low. We investigated enzymatic evaluation of GA1 using fibroblasts and MS/MS. After loading substrates, lysine, 2-aminoadipate (2AA), or GA, in fibroblasts, and incubating for 96 h, glutarylcarnitine (C5DC) levels in the media were measured. A significant increase of C5DC was observed in GA1 patients, irrespective of substrates added. 2AA showed the largest difference between patients and controls (p  = 0.0004). Results suggested enzymatic evaluation of GA1 is useful under appropriate culture conditions.
Keywords: Glutaric academia type1; GA1; Glutarylcarnitine; C5DC; Acylcarnitine; Electrospray ionization/tandem mass spectrometry; MS/MS; Fibroblasts; In vitro probe assay; Lysine; Aminoadipate; Glutaric acid;

Benzodiazepines and their pharmacologically related drugs, zolpidem and zopiclone are widely prescribed as safe drugs, but these drugs are also abused in cases of crime, suicide and drug-facilitated sexual assault. We developed a rapid and quantitative screening method for 43 benzodiazepines, their metabolites, zolpidem and zopiclone in human plasma by liquid chromatography/mass spectrometry with a small particle column. All drugs were successfully separated within 12 min using combined scan and selected ion recording (SIR) mode. The calibration curves of most drugs were linear in the concentration range 0.5–250 ng/mL with correlation coefficients exceeding 0.99. Within-day precisions (RSD, %) of this method were 1.8–15.6% (10 ng/mL) and 0.6–10.1% (100 ng/mL) and between-day precisions (RSD, %) were 1.6–16.9% (10 ng/mL) and 0.6–16.7% (100 ng/mL). The average recoveries were 70.1% (10 ng/mL) and 87.1% (100 ng/mL). The limit of detection ranged from 0.2 to 8.0 ng/mL in 37 drugs and was below 0.2 ng/mL in 6 drugs. The established method is sensitive and rapid, thus it should be useful in forensic and clinical toxicological analyses.
Keywords: LC/MS; UPLC; Benzodiazepines; Focus™; Drug screening;

Analysis of acetylene in blood and urine using cryogenic gas chromatography–mass spectrometry by Masayuki Kashiwagi; Kenji Hara; Hiroshi Fujii; Mitsuyoshi Kageura; Mutsuo Takamoto; Aya Matsusue; Tomoko Sugimura; Shin-ichi Kubo (2658-2661).
A method for quantitative analysis of acetylene in blood and urine samples was investigated. Using cryogenic gas chromatography–mass spectrometry (GC–MS), acetylene was measured with isobutane as the internal standard in the headspace method, which revealed a linear response over the entire composite range with an excellent correlation coefficient, both in blood (R  = 0.9968, range = 5.39–43.1 μg/ml) and urine (R  = 0.9972, range = 2.16–10.8 μg/ml). The coefficients of variation (CV) for blood ranged from 2.62 to 11.6% for intra-day and 4.55 to 10.4% for inter-day. The CV for urine ranged from 2.38 to 3.10% for intra-day and 4.83 to 11.0% for inter-day. The recovery rate as an index of accuracy ranged from 83 to 111%. The present method showed good reliability, and is also simple and rapid. In actual samples from a charred cadaver due to acetylene explosion, the measured concentrations of acetylene by this method were 21.5 μg/ml for femoral vein blood, 17.9 μg/ml for right atrial blood, 25.5 μg/ml for left atrial blood and 7.49 μg/ml for urine. Quantification of acetylene provides important information, because the acetylene concentration is a vital reaction or sign. For example, when acetylene is filled in a closed space and then explodes, in antemortem explosion, the blood acetylene concentration of the cadaver might be significant. On the other hand, in postmortem explosion, acetylene is not detected in blood. Furthermore, when several victims are involved in one explosion, comparison of the sample concentrations can also provide useful information to establish the conditions at the accident scene; therefore, the present method is useful in forensics.
Keywords: Acetylene; GC–MS; Quantification; Isobutane; Cryogenic;