Journal of Chromatography B (v.823, #1)

Preface by Seiji Yamaguchi; Toshimitsu Niwa; Akira Shimizu (1).

Differential chemical diagnosis of primary hyperoxaluria type II by Yoshito Inoue; Toshihiro Shinka; Morimasa Ohse; Tomiko Kuhara (2-6).
We established a separation method for the optical isomers of glyceric acid in urine by modifying the derivatization steps of the procedure used for the screening and diagnosis. The trimethylsilyl derivatization step in the mass screening procedure was replaced by O-acetyl-(+)-2-butylation, and the samples were analyzed under equivalent GC/MS conditions by capillary gas chromatography on a DB-5MS column. This method can be applied to cases that show a high urinary concentration of glyceric acid to obtain a differential diagnosis of primary hyperoxaluria type II and d-glyceric aciduria easily. l-Glyceric acid was also isolated from the urine of healthy controls as one of the main peaks.
Keywords: Primary hyperoxaluria type II; Urinary glyceric acid; Separation of enantiomers; O-Acetylated (+)-2-butyl ester;

We developed a simple and sensitive stable-isotope dilution method for the quantification of 3-hydroxyglutaric acid (3HGA) and glutaric acid (GA) in body fluids. In our method, tert-butyldimethylsilyl (tBDMS) derivatives of 3HGA and GA were measured with a conventional electron-impact ionization (EI) mode in gas chromatography–mass spectrometry (GC–MS). The control values for 3HGA in nmol/ml were 0.15 ± 0.08 (serum; n  = 10) and 0.07 ± 0.03 (CSF; n  = 10). In addition, glutarylcarnitine and free carnitine were quantified by electrospray tandem mass spectrometry. Using these methods, we monitored 3HGA, GA, and glutarylcarnitine in the body fluids of three patients with glutaric aciduria type 1 found during newborn screening. None of the patients had experienced neurological strokes, which are possibly caused by the accumulation of 3HGA, at 15–24 months of age under a disease-specific treatment, including carnitine supplementation. Our data showed that 3HGA levels were relatively high in some serum samples with lower glutarylcarnitine and carnitine levels, suggesting that carnitine supplementation may play a role in preventing the accumulation of 3HGA in patients with this disease.
Keywords: Glutaric acid; 3-Hydroxyglutaric acid; Newborn screening; Carnitine;

We performed prenatal diagnosis of organic acid disorders using two mass spectrometric methods; gas chromatography mass spectrometry (GC/MS) and tandem mass spectrometry (ESI/MS/MS). Of 28 cases whose amniotic fluid was tested, 11 cases were diagnosed as “affected”. All cases whose samples were diagnosed as “unaffected” were confirmed to have no symptoms or abnormalities in urinary organic acid analysis after birth. Of the 11 “affected” cases, two cases were missed by ESI/MS/MS but not by GC/MS. When the stability of metabolites in amniotic fluid was checked, it was found that acylcarnitines degraded in one week at room temperature, whereas organic acids such as methylmalonate or methylcitrate were stable for at least 14 days. Prenatal diagnosis by analysis using simultaneous two or more methods may be more reliable, though attention should be paid to sample transportation conditions.
Keywords: Prenatal diagnosis; Organic acid disorders; GC/MS; ESI/MS/MS; Stability of metabolites;

Quantification of sugar phosphate intermediates of the pentose phosphate pathway by LC–MS/MS: application to two new inherited defects of metabolism by Mirjam M.C. Wamelink; Eduard A. Struys; Jojanneke H.J. Huck; Birthe Roos; Marjo S. van der Knaap; Cornelis Jakobs; Nanda M. Verhoeven (18-25).
We describe a liquid chromatography tandem mass spectrometry (LC–MS/MS) method to quantify pentose phosphate pathway intermediates (triose-3-phosphates, tetrose-4-phosphate, pentose-5-phosphate, pentulose-5-phosphates, hexose-6-phosphates and sedoheptulose-7-phosphate (sed-7P)) in bloodspots, fibroblasts and lymphoblasts. Liquid chromatography was performed using an ion pair loaded C18 HPLC column and detection of the sugar phosphates was carried out by tandem mass spectrometry using an electron ion spray source operating in the negative mode and multiple reaction monitoring. Reference values for the pentose phosphate pathway intermediates in blood spots, fibroblasts and lymphoblasts were established. The method was applied to cells from patients affected with a deficiency of transaldolase. The transaldolase-deficient cells showed an increased concentration of sedoheptulose-7-phosphate. (Bloodspots: 5.19 and 5.43 μmol/L [0.49–3.33 μmol/L]; fibroblasts 7.43 and 26.46 μmol/mg protein [0.31–1.14 μmol/mg protein]; lymphoblasts 16.03 μmol/mg protein [0.61–2.09 μmol/mg protein].) The method was also applied to study enzymes of the pentose phosphate pathway by incubating fibroblasts or lymphoblasts homogenates with ribose-5-phosphate or 6-phosphogluconate and the subsequent analysis of the formed sugar phosphates.
Keywords: Sugar phosphate; Pentose phosphate pathway; LC–MS/MS;

Focused lipidomics by tandem mass spectrometry by Ryo Taguchi; Toshiaki Houjou; Hiroki Nakanishi; Toshiyuki Yamazaki; Mayuko Ishida; Masayoshi Imagawa; Takao Shimizu (26-36).
In this paper we performed focused analyses of phospholipids by using the data of precursor ion scanning and neutral loss scanning of their polar head groups and fatty acyl moieties for the specific search of categorical phospholipids. By using precursor ion scanning or neutral loss scanning of polar head groups in the positive ion mode, more sensitive identification were obtained than that in the negative ion mode. Precursor ion scanning of carbonic anions in the negative ion mode was also effective to identify molecular species of phospholipids having specified fatty acyl moieties. By using these analytical methods, the detection limits of individual metabolites are going up to 5–20-fold of former conventional methods. The important factor is that by focusing in some limited categories of molecules, detection limit is greatly enhanced, thus minor but important molecules can be detected. Moreover, combination of LC–MS/MS and focused scanning for head group was revealed to be useful to identify very minor molecular species in the focused class of phospholipids.
Keywords: Lipidomics; Metabolomics; Phospholipids; Tandem mass;

Detection of phosphatidylcholine oxidation products in rat heart using quadrupole time-of-flight mass spectrometry by Junko Adachi; Naoki Yoshioka; Mariko Sato; Kanako Nakagawa; Yorihiro Yamamoto; Yasuhiro Ueno (37-43).
An improved technique for the analysis of phosphatidylcholine (PC) and lyso-phosphatidylcholine (lyso-PC) oxidation products was developed using quadrupole time of flight (Q-TOF) mass spectrometry with electrospray ionization. We separated these products using an HPLC C8 column with a gradient of methanol and 10 mM aqueous ammonium acetate. Monohydroxides, oxo derivatives, and trihydroxides of palmitoyl-linoleoyl (C16:0/C18:2) PC, stearoyl-linoleoyl (C18:0/C18:2) PC, and oleoyl-linoleoyl (C18:1/C18:2) PC were detected mainly as MH+ and [M + Na]+ ions in the heart of the intact rat. Using standard synthetic PC-OH (C16:0/C18:2-OH), the lipid extract component was identified as (C16:0/C18:2-OH) PC based on the product ions of ESI–MS–MS and, the PC-OH concentration was quantitated. Four oxidatively modified 1-lyso-phosphatidylcholines (lyso-PCs) were also detected. This is the first report showing the presence of monohydroxides, oxo derivatives, and trihydroxides of (C16:0/C18:2)PC, (C18:0/C18:2)PC, and (C18:1/C18:2) PC in the rat heart.
Keywords: Phosphatidylcholine oxidation products; Q-TOF; Rat;

Stability of 5-aminolevulinic acid on dried urine filter paper for a diagnostic marker of tyrosinemia type I by Toshihiro Shinka; Morimasa Ohse; Yoshito Inoue; Tomiko Kuhara (44-46).
The chemical diagnosis of tyrosinemia type I generally involves the detection of succinylacetone (SA) in patient urine. However, 5-aminolevulinate (5ALA), which accumulates due to succinylacetone's inhibition of porphyrin synthesis, can also be used as diagnostic metabolites. Here we examined the stabilities of these markers on dried urine filter paper. After two weeks at room temperature, the succinylacetone was 10% of its original level, but over 80% of 5-aminolevulinate remained. Thus, although insufficient succinylacetone was recovered from dried urine filter paper to diagnose tyrosinemia type I, 5-aminolevulinate was readily detected, permitting the diagnosis.
Keywords: 5-Aminolevulinate; GC/MS analysis; Tyrosinemia type I; Dried urine filter paper;

We devised a sensitive and simple method to determine alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine, using gas chromatography–mass spectrometry (GC–MS). AMT and 5MeO-DIPT were extracted using an Extrelut® column with an internal standard, bupivacaine, followed by derivatization with acetic anhydride. The derivatized extract was used for GC–MS analysis of EI-SIM mode. The calibration curves of AMT and 5MeO-DIPT were linear in the concentration range from 10 to 750 ng/ml in both blood and urine samples. The method detection limit (MDL) of AMT and 5MeO-DIPT were 1 ng/ml each in whole blood and 5 ng/ml each in urine. This method should be most useful to accurately determine the presence of these drugs in blood and urine in clinical and forensic cases.
Keywords: Alpha-methyltryptamine; 5-Methoxy-N,N-diisopropyltryptamine;

Skin analysis to determine causative agent in dermal exposure to petroleum products by Yoko Hieda; Yoshio Tsujino; Haruo Takeshita (53-59).
This study evaluates the usefulness of skin analysis to determine the causative agent in cases of dermal exposure. The study consists of an animal experiment and two human cases. The petroleum components detected at high concentrations in skin samples resembled the composition of those in the corresponding petroleum products. However, the petroleum components in blood were detected at low concentrations and were a different composition. Skin is considered to be an advantageous sample to estimate the petroleum product in clinical and forensic cases of dermal exposure.
Keywords: Skin; Dermal exposure; Petroleum; Hydrocarbons; GC–MS;