Journal of Chromatography B (v.838, #1)

Preface by Yoshinao Wada; Toshimitsu Niwa; Akira Shimizu (1).

In memoriam by Makoto Yoshino (2).

Congenital disorders of glycosylation (CDG) constitute a group of diseases affecting N-linked glycosylation pathways. The classical type of CDG, now called CDG-I, results from deficiencies in the early glycosylation pathway for biosynthesis of lipid-linked oligosaccharide and its transfer to proteins in endoplasmic reticulum, while the CDG-II diseases are caused by defects in the subsequent processing steps. Mass spectrometry (MS) produced a milestone in CDG research, by localizing the CDG-I defect to the early glycosylation pathway in 1992. Currently, MS of transferrin, either by electrospray ionization or matrix-assisted laser desorption/ionization, plays the central role in laboratory screening of CDG-I. On the other hand, the glycopeptide analysis recently developed for site-specific glycans of glycoproteins allows detailed glycan analysis in a high throughput manner and will solve problems in CDG-II diagnosis. These techniques will facilitate studying CDG, a field now expanding to O-linked glycosylation and to acquired as well as inherited conditions that can affect protein glycosylation.
Keywords: Mass spectrometry; Glycosylation; N-linked oligosaccharide; CDG; Glycoprotein;

Evaluation of mutation effects on UGT1A1 activity toward 17β-estradiol using liquid chromatography–tandem mass spectrometry by Keiko Wada; Atsuko Takeuchi; Kayoko Saiki; Retno Sutomo; Hans Van Rostenberghe; Narazah Mohd Yusoff; Vichai Laosombat; Ahmad Hamim Sadewa; Norlelawati Abdul Talib; Surini Yusoff; Myeong Jin Lee; Hitoshi Ayaki; Hajime Nakamura; Masafumi Matsuo; Hisahide Nishio (9-14).
Mutations in the gene encoding UDP-glucuronosyltransferase 1A1 (UGT1A1) may reduce the glucuronidation of estradiol, bilirubin, etc. In the present study, we used a liquid chromatography–tandem mass spectrometry (LC/MS/MS) method to assay the activities of recombinant mutated UGT1A1 toward 17β-estradiol (E2), by determining its glucuronide (E2G) content. Direct evidence for glucuronide formation was provided by E2G-specific ion peaks. The UGT1A1 activities of G71R (exon 1), F83L (exon 1), I322V (exon 2) and G493R (exon 5) mutants were 24, 30, 18 and 0.6% of the normal UGT1A1 activity, respectively. In conclusion, our study showed that LC/MS/MS enabled accurate evaluation of the effects of mutations on recombinant UGT1A1 activity towards E2.
Keywords: UGT1A1; Mutation; 17β-Estradiol;

To screen cancer for specific autoantibodies, we applied the approach established by Brichory et al., who reported annexins I and II as specific antigens. Solubilized proteins from a cancer cell line (A549) were separated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by Western blotting (WB) analysis, in which the sera of individual patients were tested for primary antibodies. We found 11 positive spots on PVDF membrane using a WB/enhanced chemiluminescence detection Kit, and identified eight proteins, such as α-enolase, inosine-5′-monophosphate dehydrogenase, aldehyde dehydrogenase, 3-phosphoglycerate dehydrogenase, 3-oxoacid CoA transferase, chaperonin, peroxiredoxin 6 and triosephosphate isomerase, that reacted with these antibodies in patients’ sera using MALDI-TOF/TOF. All eight antibodies were not detected in the sera derived from lung tuberculosis and healthy controls.
Keywords: Lung adenocarcinoma-specific antibody; Two-dimensional polyacrylamide gel electrophoresis; Lung adenocarcinoma; Matrix-assisted laser desorption ionization/time-of-flight mass spectrometry;

To verify the exposure to nerve gas, a method for detecting human butyrylcholinesterase (BuChE)-nerve gas adduct was developed using LC–electrospray mass spectrometry (ESI-MS). Purified human serum BuChE was incubated with sarin, soman or VX, and the adduct was purified by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and digested in gel by treatment with chymotrypsin. The resulting peptide mixture was subjected to LC–ESI-MS. From the chymotryptic digest of untreated human BuChE, one peak corresponding to the peptide fragment containing the active center serine residue was detected on the extracted ion chromatogram at m/z 948.5, and the sequence was ascertained to be “GESAGAASVSL” by MS/MS analysis. From the chymotryptic digest of the human BuChE-sarin adduct, a singly charged peptide peak was detected on the extracted ion chromatogram at m/z 1069.5, and the sequence was ascertained to be “GEXAGAASVSL” by MS/MS analysis (X denotes isopropylmethylphosphonylated serine). The difference in molecular weight (120.0 Da) between the active center peptide fragments corresponding to the untreated BuChE and BuChE-sarin adduct was assumed to be derived from the addition of an isopropyl methylphosphonyl moiety to the serine residue. The formation of human BuChE adducts with soman, VX and an aged soman adduct was confirmed by detecting the respective active center peptide fragments using LC–ESI-MS. To apply the established method to an actual biological sample, human serum was incubated with VX, and the adduct was purified by procainamide affinity chromatography followed by SDS-PAGE. After chymotryptic in gel digestion, the ethylphosphonylated active center peptide fragment could be detected, and the structure of the residue was ascertained by LC–ESI-MS analysis.
Keywords: Nerve gas; Butyrylcholinesterase; Adduct; LC–MS; Chymotryptic digestion;

Quantification of lysophosphatidylcholines and phosphatidylcholines using liquid chromatography–tandem mass spectrometry in neonatal serum by Akihiro Takatera; Atsuko Takeuchi; Kayoko Saiki; Takeshi Morisawa; Naoki Yokoyama; Masafumi Matsuo (31-36).
We established an improved method for quantification of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) molecular species in neonatal serum using high-performance liquid chromatography coupled tandem mass spectrometry (LC–MS/MS). A multiple reaction monitoring (MRM) mode of positive ionization for MS/MS was used. The method involved purification of phospholipids by solid phase extraction (SPE) from a 20-μl minimum specimen of serum. The assayed values of authentic 16:0-LPC and 18:0-LPC showed a linear response, and our quantitative results showed high precision for the all species of PC and LPC. Then, we quantified PC and LPC in adult and neonatal serum and compared them. Day 0–1 neonatal serum 16:0-, 18:0-, 18:1-, 18:2-LPC levels were significantly lower than adult ones. All species LPC levels in the day 0–1 neonates were significantly lower than day 4–8 neonates. Day 0–1 neonatal serum 16:0/18:2-, 18:0/18:2-PC levels were significantly lower than adult ones. Our method is advantageous for precise assessments of the relationships between PCs/LPCs levels and neonatal infectious diseases.
Keywords: Lysophosphatidylcholine; Phosphatidylcholine; Neonate; Liquid chromatography–tandem mass spectrometry;

To establish a method for separating the optical isomers of lactic acid, we modified the derivatization steps in our procedure for urinary mass-screening for inborn errors of metabolism. For chiral recognition, we chose O-trifluoroacetyl-(−)-menthylation derivatization instead of our previous method, trimethylsilyl derivatization, and the samples were then analyzed under GC/MS by capillary gas chromatography on a DB-5MS column. This method can be used to follow-up the condition of a patient with short bowel syndrome and to prevent onset and/or seizure. d-Lactic acid was also isolated from the urine of healthy controls as one of the main peaks in the chromatogram.
Keywords: Short bowel syndrome; Urinary d-lactic acid; Separation of enantiomers; O-trifluoroacetylated (−)-menthyl ester;

A new method, using sudan I as internal standard to determine the content of lycopene or β-carotene in samples, was developed. According to UV–vis absorption spectra, sudan I, lycopene and β-carotene all had absorption peaks at 450 nm. They could be separated absolutely by high-performance liquid chromatography (HPLC) with retention time of 2.7, 6.6 and 10.1 min, respectively. The related equations between sudan I and lycopene or β-carotene content were obtained and verified by determining the content of lycopene or β-carotene in Blakeslea trispora cells. The relative error was below 1.4% for determining lycopene content and below 1.9% for β-carotene. Intra-day variability for lycopene determination was less than 3.4% and for β-carotene was less than 1.4%. The mean recovery of lycopene or β-carotene was 96.1 and 97.9%, respectively.
Keywords: Lycopene; β-Carotene; Sudan I; HPLC; Internal standard;

A simple HPLC method using column-switching and ultraviolet detection was developed for the simultaneous determination of baicalin (BA), rhein (RH) and berberine (BE) in rat plasma. Plasma samples were injected directly onto a Capcell Pak MF C8 column (150 mm × 4.6 mm i.d.) to remove protein and to be pre-separated by an isocratic elution using 50 mmol/L phosphate sodium (pH 6.85)–acetonitrile (10:1, v/v). After the drug-containing fractions were transferred to a Kromasil C18 column (150 mm × 4.6 mm i.d.) by a valve switching step, the valve position was switched back and the main separation was performed by an isocratic elution using triethylamine adjusted 20 mmol/L phosphoric acid (pH 6.78)–acetonitrile (4:1, v/v). The flow rate was always 1.0 mL/min. The calibration curve showed excellent linear relationship (r  ≥ 0.9997) over the concentration range of 0.4–7.9 μg/mL for baicalin, 0.2–7.8 μg/mL for rhein and 0.4–7.7 μg/mL for berberine in rat plasma. The intra- and inter-day assay precisions (R.S.D.) of three analytes were in the range of 0.34–4.3% and the accuracies were between 98.0% and 102.4%. Their recoveries were all greater than 95%. The method was successfully applied to the multi-constituents plasma concentration–time curve study after oral administration of a traditional Chinese medicine prescription Xiexin-Tang in rats.
Keywords: HPLC; Plasma; Column-switching; Traditional Chinese medicine; Baicalin; Rhein; Berberine;

Enantioselective recognition of 2,3-benzodiazepin-4-one derivatives with anticonvulsant activity on several polysaccharide chiral stationary phases by Maria Luisa Calabrò; Daniela Raneri; Silvana Tommasini; Rita Ficarra; Stefano Alcaro; Andrea Gallelli; Nicola Micale; Maria Zappalà; Paola Ficarra (56-62).
The retention behaviour of racemic 1-(4-aminophenyl)-1,2,3,5-tetrahydro-7,8-methylendioxy-4H-2,3-benzodiazepin-4-one derivatives with anticonvulsant activity on several chiral stationary phases was investigated. The selective performances of six polysaccharide phases, namely, Chiralcel OA, OD, OF, OG, OJ and Chiralpak AD were studied and normal phase HPLC methods were optimized to separate the enantiomeric forms of this class of compounds. The chiral recognition mechanism between the analytes and the chiral selectors was discussed. A molecular modeling study was carried out with the aim to explore the enantioselective molecular recognition process with the Chiralcel OG stationary phase.
Keywords: Chiral separation; Benzodiazepines; Polysaccharide chiral stationary phases; Enantiomer separation; Docking;

Application of liquid chromatography–mass spectrometry to the investigation of poisoning by Oenanthe crocata by Geoffrey C. Kite; Charlotte A. Stoneham; Nigel C. Veitch; Bridget K. Stein; Katherine E. Whitwell (63-70).
Liquid chromatography–mass spectrometry (LC–MS) analysis of methanol extracts of Oenanthe crocata roots revealed that oenanthotoxin co-eluted with another major polyalkyne, 2,3-dihydro-oenanthotoxin, using the existing high performance liquid chromatography (HPLC) method (isocratic elution from C18 with aqueous methanol) for investigating Oenanthe poisoning. Positive ES or APCI gave [(M + H)–H2O]+ and its methanol adduct as major ion species for oenanthotoxin, whereas 2,3-dihydro-oenanthotoxin formed [M + H]+ and its methanol adduct. The two polyalkynes could be chromatographically resolved on C18 by gradient elution with aqueous acetonitrile. This provides superior analysis for oenanthotoxin using HPLC with photodiode array (PDA) detection alone, but for LC–MS/MS aqueous acetonitrile was less suitable due to poor ionisation and, with APCI, an increase in the relative abundance of a [M-1]+ species, which could confuse compound assignment. HPLC–PDA and LC–MS/MS methods using an aqueous acetonitrile or aqueous methanol mobile phase, respectively, were successful when applied to the analysis of the stomach contents of a pony suspected to have eaten O. crocata. Relevant product ion spectra, by ion trap MS/MS, accurate mass data and complete sets of 1H and 13C NMR spectral assignments are given for the two compounds.
Keywords: Oenanthe crocata; Oenanthotoxin; Poisoning; Liquid chromatography–mass spectrometry;