Journal of Chromatography B (v.776, #1)

Preface (1).

Application of gas chromatography–surface ionization organic mass spectrometry to forensic toxicology by Akira Ishii; Kanako Watanabe-Suzuki; Hiroshi Seno; Osamu Suzuki; Yoshinao Katsumata (3-14).
Surface ionization (SI), which consists in the formation of positive and negative ions along the course of thermal desorption of particles from a solid surface, was first applied as a detector for gas chromatography (GC), GC-surface ionization detection (SID); we developed many new sensitive methods for the determination of abused and other drugs by GC-SID. Recently, Fujii has devised a combination of SI and a quadrupole mass spectrometer and named this system a surface ionization organic mass spectrometer (SIOMS), which is highly selective and sensitive for organic compounds containing tertiary amino groups. We have tried to apply this mass spectrometer to forensic toxicological study; so far we have succeeded in determining important drugs-of-abuse and toxic compounds, such as phencyclidine (PCP), pethidine, pentazocine, MPTP and its derivatives from human body fluids with high sensitivity and selectivity. In this review, we describe our recent studies on the application of GC–SIOMS to forensic toxicology.
Keywords: Phencyclidine; Pethidine; Pentazocine;

The detection and identification of protein variants and abnormally increased modified proteins are important for clinical diagnosis. We applied soft ionization mass spectrometry (MS) to analyze proteins in blood and tissues from various patients. Over the past 8 years, we diagnosed 132 cases (55 kinds) of variant proteins including hemoglobin (Hb), transthyretin (TTR), and Cu/Zn-superoxide dismutase (SOD-1), using MS as the leading technology. Of these variants, eight were new, and nine were the first cases in Japan. Some abnormal Hb cause diseases, and most of them cause erroneous levels of glycated Hb, HbA1c, i.e., a popular index of diabetes. Most of the variant TTR causes amyloidotic polyneuropathy. Variant SOD-1 causes amyotrophic lateral sclerosis. We first showed that immunoprecipitation by a specific antiserum is a reliable and simple method to prepare protein from sera and tissues for analysis by matrix-assisted laser desorption time-of-flight MS, and liquid chromatography–electrospray ionization MS (LC–ESI-MS). The use of this technology has become widespread. Using an immunoprecipitated target protein and LC–ESI-MS, we showed that the ratios of tetra-, di- and a-sialo-transferrin from two cases of congenital glycoprotein deficient syndrome were clearly distinguishable from those of control samples. We first reported a unique modified form of TTR, that is, S-sulfonated TTR, which increased markedly and specifically in three cases with molibdenum cofactor deficiency. We proposed that S-sulfonated TTR is a useful marker for screening this disease. ESI-MS was successfully used for the accurate determination of HbA1c, and we clarified the extent of discrepancies between the HbA1c value measured by conventional methods and the accurate values for samples containing various Hb variants determined by the MS method.
Keywords: Protein variants; Protein adducts;

A new technique for accurate mass measurement utilizing multiple sprayer nano-electrospray ionization mass spectrometry (nano-ESI-MS) combined with nano-scale high-performance liquid chromatography (nano-HPLC) on a magnetic sector instrument is described. Both metal-coated glass capillaries and fused-silica capillaries were used as nano-ESI sprayers. A metal-coated glass capillary was used for the introduction of the Ref. compound solution, and a metal-coated fused-silica capillary was used for connection to the nano-HPLC column. By shifting each sprayer’s position relative to the sampling orifice, spectra were obtained of both the sample components as eluted from the column and reference compounds. Several standard compounds were examined and satisfactory accurate masses were obtained. Problems arising from differences in ionization efficiency between the sample and reference compounds were not observed.

Newborn mass screening and selective screening using electrospray tandem mass spectrometry in Japan by Yosuke Shigematsu; Satoko Hirano; Ikue Hata; Yukie Tanaka; Masakatsu Sudo; Nobuo Sakura; Tsuyoshi Tajima; Seiji Yamaguchi (39-48).
Electrospray tandem mass spectrometry was applied to detect a series of inherited metabolic disorders during a newborn-screening pilot study and a selective screening in Japan. In our mass screening of 102 200 newborns, five patients with propionic acidemia, two with methylmalonic acidemia, two with medium-chain acyl-CoA dehydrogenase deficiency, three with citrullinemia type II, and one with phenylketonuria were identified. In a selective screening of 164 patients with symptoms mainly related to hypoglycemia and/or hyperammonemia, 12 with fatty acid oxidation disorders and six with other disorders were found. The results indicated the importance of newborn screening using this technology in Japan.

The purpose of this study was to develop a simple and accurate analytical method to determine amino acids in urine samples. The developed method involves the employment of an extract derivatization technique together with gas chromatography–mass spectrometry (GC–MS). Urine samples (300 μl) and an internal standard (10 μl) were placed in a screw tube. Ethylchloroformate (50 μl), methanol–pyridine (500 μl, 4:1, v/v) and chloroform (1 ml) were added to the tube. The organic layer (1 μl) was injected to a GC–MS system. In this proposed method, the amino acids in urine were derivatized during an extraction, and the analytes were then injected to GC–MS without an evaporation of the organic solvent extracted. Sample preparation was only required for ca. 5 min. The 15 amino acids (alanine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tyrosine, tryptophan, valine) quantitatively determined in this proposed method. However, threonine, serine, asparagine, glutamine, arginine were not derivatized using any tested derivatizing reagent. The calibration curves showed linearity in the range of 1.0–300 μg/ml for each amino acid in urine. The correlation coefficients of the calibration curves of the tested amino acids were from 0.966 to 0.998. The limit of detection in urine was 0.5 μg/ml except for aspartic acid. This proposed method demonstrated substantial accuracy for detection of normal levels. This proposed method was limited for the determination of 15 amino acids in urine. However, the sample preparation was simple and rapid, and this method is suitable for a routine analysis of amino acids in urine.
Keywords: Amino acids;

We describe the rapid and sensitive detection of 4-hydroxybutyric acid, which is a marker compound for succinic semialdehyde dehydrogenase (SSADH) deficiency. Urinary 4-hydroxybutyric acid and 3,4-dihydroxybutyric acid were targeted, quantified by gas chromatography–mass spectrometry after simplified urease digestion in which lactone formation from γ-hydroxy acids is minimized. The recovery of 4-hydroxybutyric acid using this method was over 93%. 2,2-Dimethylsuccinic acid was used as an internal standard. The detection limit of this method was 1 nmol ml−1 for both 4-hydroxybutyric acid and 3,4-dihydroxybutyric acid. The urinary concentrations of 4-hydroxybutyric acid and of 3,4-dihydroxybutyric acid from the patient with an SSADH deficiency were 880–3628 mmol mol−1 creatinine (control; 3.3±3.3 mmol mol−1 creatinine) and 810–1366 mmol mol−1 creatinine (control; 67.4±56.2 mmol mol−1 creatinine), respectively. The simplified urease digestion of urine is very useful for quantifying 4-hydroxybutyric acid and its related compounds in patients with 4-hydroxybutyric aciduria.
Keywords: 4-Hydroxybutyric acid;

Two cases of benign methylmalonic aciduria detected during a pilot study of neonatal urine screening by Toshihiro Shinka; Yoshito Inoue; Makoto Yoshino; Hiroaki Kakinuma; Hiroaki Takahashi; Tomiko Kuhara (65-70).
Two cases of benign methylmalonic aciduria (MMAuria) were found among 9780 neonatal screenings using the previously described screening method consisting of urease digestion, ethanol deproteinization and gas chromatography–mass spectrometry. Combining this screening method with the stable isotope dilution technique showed very specific and sensitive measurements of methylmalonic acid in urine. The concentrations of urinary methylmalonic acid were measured at several ages. The levels of urinary methylmalonic acid in two patients varied from 0.27 to 3.04 mol/mol creatinine (control<0.01 mol/mol creatinine). Methylcitrate and homocystine were not increased in the patient’s urine or blood. Blood propionylcarnitine was also at normal levels. The urinary methylmalonate excretions were decreased to the levels of about 50% of the start point after vitamin B12 treatment in one patient, but the other patient showed no change. No clinical abnormalities were observed during these periods.

Propionic acidemia is one of the most frequent inborn errors of metabolism caused by a deficiency of propionyl-CoA carboxylase. Methylcitric acid, a key indicator of this disorder, is increased in amniotic fluid when a fetus is affected. Therefore, the direct chemical analysis of cell-free amniotic fluid for methylcitric acid, using stable isotope dilution gas chromatography–mass spectrometry, was carried out for the prenatal diagnosis of propionic acidemia. We developed a simple, highly sensitive, and accurate method for quantitation of this polar methylcitric acid in amniotic fluids by applying a simplified urease pretreatment which we devised earlier for urine. As the recovery of methylcitric acid from amniotic fluid was as high as 91% with a coefficient of variation lower than 3% in this procedure, only 0.02 ml of sample was required for the analysis of the affected fetus. This new procedure takes 1 h for sample pretreatment, including derivatization, and 15 min for GC–MS measurement and provides final results within 1.5 h.
Keywords: Methylcitric acid;

In order to confirm that diisopropylfluorophosphate (DFP) phosphorylates the active site serine residue in α-chymotrypsin, a peptide containing the phosphorylated active site was analyzed by liquid chromatography (LC)–electrospray mass spectrometry (ESI-MS). After reduction with dithiothreitol and subsequent alkylation with acrylamide, α-chymotrypsin was digested by treatment with trypsin. Tryptic digest was subjected to LC–ESI-MS. Nearly all the peptide fragments were identified by comparison with fragments predicted from as tryptic digest of α-chymotrypsin. From the tryptic digest of native α-chymotrypsin, a doubly protonated peptide peak which corresponded to the peptide fragment containing the active site serine residue was detected on a selected ion chromatogram at m/z 1265.0, and the sequence was determined to be “DAMICAGASGVSSCMGDSGGPLVCK”. From the tryptic digest of DFP-inhibited α-chymotrypsin, the doubly protonated peptide peak was detected on a selected ion chromatogram at m/z 1347.0. The difference in mass number (82 in a doubly charged ion) of active site peptide fragments between the native and DFP inhibited α-chymotrypsins was assumed to be the result of phosphorylation of the serine residue with a diisopropylphosphoryl moiety. A total of +164 Da mass shifts of y-series fragment ions from the y8 to y21 positions in the active site peptide of the DFP inhibited α-chymotrypsin was observed, in comparison with the native α-chymotrypsin. Thus, the phosphorylation site in α-chymotrypsin could be unequivocally identified to be at the serine residue which is located at position 47, from the N-terminus of the α-chymotrypsin C-chain.
Keywords: Serine protease; Organophosphorus;

Catalogue of soluble proteins in the human vitreous humor: comparison between diabetic retinopathy and macular hole by Toyofumi Nakanishi; Reiko Koyama; Tsunehiko Ikeda; Akira Shimizu (89-100).
Two-dimensional gel electrophoresis and mass spectrometry were used to make a catalogue of soluble proteins in the human vitreous humor (VH). Fifty-one different proteins were identified on silver-stained two-dimensional (2D) gel patterns with VH proteins obtained from diabetic retinopathy and macular hole. Thirty of these have not been listed in the reported 2D profiles of plasma. Immunoglobulin (Ig), α1-antitrypsin, α2-HS glycoprotein,and complement C4 fragment showed stronger spots in VH with diabetic retinopathy patient samples than those with macular hole. Pigment epithelium-derived factor, a potent inhibitor of angiogenesis in the cornea and vitreous, was clearly detected in VH with diabetes. It is impressive that the inhibitor increases in the vitreous with proliferative angiogenesis.
Keywords: Proteins;

Quantitative analysis of IgA1 binding protein prepared from human serum by hypoglycosylated IgA1/Sepharose affinity chromatography by Ikuko Nakamura; Hitoo Iwase; Yukichi Ohba; Yoshiyuki Hiki; Tadashi Katsumata; Yutaka Kobayashi (101-106).
The binding protein to a hypoglycosylated IgA1/Sepharose (IgA1-BP) could be prepared from human sera. IgG was a major component in the IgA1-BP. A Protein A column was used to remove the IgG; however, about half of the IgA1-BP was passed from the column [Biochem. Biophys. Res. Commun., 264 (1999) 424]. Quantitative analysis of the passed fraction (PAP) by laser nepherometry indicated that it was composed of a fairly large amount of IgA, IgM and complement C3 besides IgG. The relative content of IgG:IgA:IgM:C3:C4 was 25:10:41:22:2 in the PAP fraction. Meanwhile, the Protein A bound-fraction was essentially composed of IgG (78%) and IgM (19%). The total amount of IgA1-BP was not different between the sera from IgA nephropathy patients and other nephropathy patients. With respect to the IgA content in the IgA1-BP from IgA nephropathy patients, it was significantly higher than that from other nephropathy patients. It was found that the IgA1-BP from some IgA nephropathy patients contained a few micrograms of aberrant IgA per ml of serum. Thus, the obtained results suggested the preferential deposition of the self-aggregated IgA composed of hypoglycosylated IgA1 and co-deposition of IgG, IgM and C3 in the glomeruli in an IgA nephropathy patient.
Keywords: Immunoglobulins; Proteins;

Liquid chromatographic–mass spectrometric determination of haloperidol and its metabolites in human plasma and urine by Tetsuya Arinobu; Hideki Hattori; Masae Iwai; Akira Ishii; Takeshi Kumazawa; Osamu Suzuki; Hiroshi Seno (107-113).
Haloperidol and its two metabolites, reduced haloperidol and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP) in human plasma and urine were analyzed by HPLC–MS using a new polymer column (MSpak GF-310), which enabled direct injection of crude biological samples without pretreatment. Recoveries of haloperidol and reduced haloperidol spiked into plasma were 64.4–76.1% and 46.8–50.2%, respectively; those for urine were 87.3–99.4% and 94.2–98.5%, respectively; those of CPHP for both samples were not less than 92.7%. The regression equations for haloperidol, reduced haloperidol and CPHP showed good linearity in the ranges of 10–800, 15–800 and 400–800 ng/ml, respectively, for both plasma and urine. Their detection limits were 5, 10 and 300 ng/ml, respectively, for both samples. Thus, the present method was sensitive enough for detection and determination of high therapeutic and toxic levels for haloperidol and its metabolites present in biological samples.
Keywords: Haloperidol;

Gaucher disease is a glycolipid storage disorder characterized by the accumulation of glucosylceramide. Using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE–MALDI-TOF-MS), we analyzed sphingolipids in pericardial fluid, peritoneal fluid, and serum from two patients with Gaucher disease. Crude lipids were extracted from 1 ml each of pericardial fluid, peritoneal fluid, and serum with chloroform and methanol. After mild alkaline treatment of the crude lipids, a sphingolipid fraction was prepared and analyzed by DE–MALDI-TOF-MS. The results were as follows: (a) in all the specimens, peaks of ceramide monohexoside and sphingomyelin were detected in both the controls and Gaucher disease patients; (b) in pericardial fluid, peritoneal fluid, and serum, the ceramide monohexoside/sphingomyelin ratio was increased in the Gaucher disease patients compared with in the controls. It was indicated that the accumulation of ceramide monohexoside in such samples from Gaucher disease patients can be easily detected with this DE–MALDI-TOF-MS method.
Keywords: Sphingolipids;

Rapid and simple quantitative assay method for diastereomeric flurbiprofen glucuronides in the incubation mixture by Nariyasu Mano; Ayako Nikaido; Takashi Narui; Daisuke Yamasaki; Junichi Goto (125-131).
Acyl glucuronides of nonsteroidal anti-inflammatory drugs having a chiral center are known to be chemically very active and form covalently bound adducts with proteins, such as human serum albumin, which may be the cause of hypersensitive reactions. Hepatic acyl glucuronosyltransferase catalyzes the transformation of α-aryl propionates into these diastereoisomeric acyl glucuronides, and, hence, its activity needs to be characterized. From this point of view, we developed a rapid, accurate and reproducible analytical method for the separation and determination of diastereoisomeric glucuronides of flurbiprofen, one of the nonsteroidal anti-inflammatory drugs, in the incubation mixture of the hepatic microsomal preparation by high-performance liquid chromatography with a simple column-switching technique for deproteinization. The glucuronides were separated on a TSKgel ODS-80Ts column with 20 mM ammonium acetate buffer (pH 5.6)–ethanol–acetonitrile as the mobile phase and monitored with a UV detector at 246 nm. The detection limit of the proposed method was 600 fmol/injection at a signal-to-noise ratio of 10. The validation results indicated that this method would be very useful for the determination of diastereomeric acyl glucuronides formed from flurbiprofen in an incubation mixture.
Keywords: Flurbiprofen glucuronides;

Gas chromatographic–mass spectrometric determination of erythrocyte 3-deoxyglucosone in diabetic patients by Saori Tsukushi; Mitsuharu Kajita; Sakurako Nakamura; Toshimitsu Niwa (133-137).
To determine if the erythrocyte levels of 3-deoxyglucosone (3-DG) are increased in diabetic patients, and if they correlate with glycemic status, they were measured in diabetic patients without renal disease as well as in healthy subjects. The erythrocyte levels of 3-DG were measured by a selected ion monitoring method of gas chromatography–chemical ionization mass spectrometry using [13C6]-3-DG as an internal standard. The erythrocyte levels of 3-DG were significantly higher in diabetic patients than in healthy subjects. The erythrocyte concentration of 3-DG was significantly and positively correlated with HbA1c (r=0.84, P<0.001). However, no significant correlation could be found between erythrocyte 3-DG and age, onset age of diabetes, or duration of diabetes in our group of diabetic patients. In diabetes, the production of 3-DG in the erythrocytes is increased via the polyol pathway and/or the Maillard reaction due to hyperglycemia.
Keywords: 3-Deoxyglucosone;