Journal of Chromatography B (v.870, #2)

Preface by Yosuke Shigematsu; Toshimitsu Niwa; Akira Shimizu (145).

In memory of my teacher, Professor Akira Shimizu by Toyofumi Nakanishi (146-147).

By the development of soft ionization such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), mass spectrometry (MS) has become an indispensable technique to analyze proteins. The combination of protein separation and identification such as two-dimensional gel electrophoresis and MS, surface-enhanced laser desorption/ionization-MS, liquid chromatography/MS, and capillary electrophoresis/MS has been successfully applied for proteome analysis of urine and plasma to discover biomarkers of kidney diseases. Some urinary proteins and their proteolytic fragments have been identified as biomarker candidates for kidney diseases. This article reviews recent advances in the application of proteomics using MS to discover biomarkers for kidney diseases.
Keywords: Mass spectrometry; Biomarker; Kidney disease; Proteome;

Determination of 3-hydroxyisovalerylcarnitine and other acylcarnitine levels using liquid chromatography–tandem mass spectrometry in serum and urine of a patient with multiple carboxylase deficiency by Yasuhiro Maeda; Tetsuya Ito; Hironori Ohmi; Kyoko Yokoi; Yoko Nakajima; Akihito Ueta; Yukihisa Kurono; Hajime Togari; Naruji Sugiyama (154-159).
Due to its increased concentration in blood, 3-hydroxyisovalerylcarnitine (C5OH-I) is an important indicator for the diagnosis of organic acidemias in newborns. However, C5OH-I has not been used as a standard in tandem mass spectrometric (MS/MS) assays because its isolation is difficult. We developed a new synthesis of C5OH-I and investigated its behavior by MS/MS. A method using the multiple reaction monitoring (MRM) mode of MS/MS with HPLC was developed which provides high accuracy, precision and reproducibility. Acylcarnitine profiles in the serum and urine of a patient with multiple carboxylase deficiency (MCD) showed increased levels compared to a healthy patient.
Keywords: Acylcarnitine; 3-Hydroxyisovalerylcarnitine; 3-Methylcrotonylcarnitine; Tiglylcarnitine; Multiple carboxylase deficiency; Holocarboxylase synthetase deficiency; Newborn screening; Tandem mass spectrometry; LC–MS/MS;

Propionic acidemia is a frequent inborn error of metabolism. Methylcitric acid, a key indicator of propionic acidemia, increases in the amniotic fluid of affected fetuses. For prenatal diagnosis, the methylcitric acid in amniotic fluid can be measured by stable-isotope dilution GC/MS. Here, we quantified this indicator in samples of amniotic fluid that had been dried on filter paper and transported at ambient temperatures, and compared the results with data obtained from the original amniotic fluid. We then used the filter-paper method to screen at-risk fetuses and obtained a clear-cut diagnosis in each case.
Keywords: Dried amniotic fluid on filter paper; Propionic acidemia; Prenatal diagnosis; Methylcitric acid; GC/MS;

Simultaneous determination of prednisolone, prednisone, cortisol, and cortisone in plasma by GC–MS: Estimating unbound prednisolone concentration in patients with nephrotic syndrome during oral prednisolone therapy by Hiromi Shibasaki; Hideaki Nakayama; Takashi Furuta; Yasuji Kasuya; Mari Tsuchiya; Akinori Soejima; Akira Yamada; Toshihiko Nagasawa (164-169).
Individual variability of the pharmacokinetics of prednisolone based on the unbound concentration in plasma is of significant clinical consideration. The unbound concentrations of prednisolone were measured in 10 patients with nephrotic syndrome, two patients with systemic lupus erythematosus, and one patient with dermatomyositis by examining protein bindings of prednisolone on one or more occasions during prednisolone treatment. In this study, plasma concentrations of prednisolone, prednisone, cortisol, and cortisone were simultaneously analyzed by GC–MS by using stable isotope-labeled internal standards. Equilibrium dialysis was employed to accurately estimate the unbound fractions of prednisolone in plasma. The unbound fraction of prednisolone changed depending on plasma total prednisolone concentration and plasma albumin concentration. The unbound fraction of prednisolone (Y) is calculated: Y  = (−0.0101x′ + 0.0736) x  + 10.23, where x′ is the plasma albumin concentration and x is the total prednisolone concentration. The estimated concentrations of unbound prednisolone by using the above equation were in good agreement with the measured concentrations of unbound prednisolone. Since the protein binding of prednisolone did not change in the presence of prednisone (114.0 ng/ml), it appeared that prednisone produced from the therapeutic dose of prednisolone did not affect the unbound fraction of prednisolone.
Keywords: Prednisolone; GC–MS; Prednisolone-13C4; Prednisone-13C4; Cortisol-2H5; Cortisone-2H5; BMD-monoHFB derivatives; Protein binding; Nephrotic syndrome; Albumin;

Niemann-Pick disease types A and C, and Gaucher disease are glycolipid storage disorders characterized by the systemic deposition of glycosphingolipids, i.e., sphingomyelin in Niemann-Pick disease types A and C tissues and glucosylceramide in Gaucher disease ones, respectively. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS), we analyzed the sphingolipids in liver and spleen specimens from patients with Niemann-Pick disease types A and C, and Gaucher disease. Crude lipids were extracted from tissue containing 5 mg protein with chloroform and methanol. After mild alkaline treatment of the crude lipids, a sphingolipid fraction was prepared and analyzed by MALDI-TOF/MS. The results were as follows: (a) ion peaks with m/z values corresponding to different sphingomyelin and ceramide monohexoside (CMH) species were clearly detected. (b) With sphingosylphosphorylcholine as the internal standard for quantification of sphingomyelin and CMH, the relative peak heights of sphingomyelin and CMH were calculated and plotted versus their contents. The relative peak heights of sphingomyelin and CMH showed linearity between 50 and 1500 ng sphingomyelin content, and between 5 and 150 ng CMH content, respectively. (c) Quantitative analysis revealed the accumulation of sphingomyelin in the liver and spleen specimens from the patients with Niemann-Pick disease types A and C. Striking accumulation of CMH was also detected in the liver and spleen specimens from the patients with Gaucher disease. This investigation indicated that accumulated sphingomyelin and CMH in small amounts of tissues from sphingolipidosis patients can be detected quantatively with the MALDI-TOF/MS method. This method will be useful not only for the diagnosis but also for biochemical pathophysiology evaluation of patients with various sphingolipidosis.
Keywords: Mass spectrometry; Niemann-Pick disease; Gaucher disease; Sphingolipid;

This work characterized the metabolism disorders of acute liver failure (ALF) induced by carbon tetrachloride (CCl4) in a mouse model with different dosage of intoxication (100, 500 and 1000 mg/kg). Metabolic profiles of mice plasma were detected by gas chromatography/mass spectrometry (GC/MS) after chemical derivatization. Here an effective information-extracting approach was implemented on the basis of partial least square regression analysis (PLS-RA). PLS modeling was achieved with two kinds of Y-vectors for the acquired metabonomics data and eight metabolites with different changing behaviors were selected. ALF of mice induced by CCl4 was characterized by the elevation of glutamate, citrate, serine and threonine, as well as the decrease of α-glycerophosphate, docosahexaenoic acid, palmitic acid and oleic acid in plasma. The difference in the concentrations of serine, threonine, palmitic acid and oleic acid remained insignificant between the control and 100 mg/kg groups, while significant distinction appeared when comparing the control and two higher dosed groups. The underlying regulation of CCl4-perturbed metabolic pathways was discussed according to the selected metabolites. The present study demonstrated a great potential of PLS-RA in exploiting a comprehensive metabolic effects of CCl4 intoxication and its efficient capability to reveal the hepatotoxic mechanism of ALF induced by reactive oxygen species (ROS).
Keywords: Metabonomics; PLS-RA; GC/MS; Carbon tetrachloride; Reactive oxygen species; Acute liver failure;

Atmospheric pressure photoionization (APPI) as an interface for the high-performance liquid chromatography (HPLC)–tandem mass spectrometry (MS/MS) system was employed for the direct determination of 17alpha-ethinylestradiol (EE2) in the incubation mixtures to support in vitro hepatic clearance studies. For the APPI source, the radical cation of the analyte via charge exchange with the dopant radical cation was used for the detection of EE2 in the positive ion mode. It was demonstrated that the major signals of EE2 in the acetonitrile/water mobile phase were substantially increased by replacing toluene with anisole as the dopant. The effects of several experimental conditions on the photoionization efficiency of EE2 in the dopant-assisted APPI source were explored. Electrospray ionization (ESI) source was also suitable for the analysis of the analyte; however, ESI required a derivatization step prior to analysis. The applicability of the proposed HPLC–APPI–MS/MS approach following a protein precipitation procedure for the determination of EE2 at low nano-mole levels was examined with respect to assay specificity and linearity. The assay results obtained by both HPLC–APPI–MS/MS and HPLC–ESI–MS/MS methods were in good agreement.
Keywords: HPLC–MS/MS; Electrospray ionization; Atmospheric pressure photoionization; 17alpha-ethinylestradiol;

In this paper, the possibility of using a multiple ionization mode approach of GC/MS was developed for the simultaneous hair testing of common drugs of abuse in Asia, including amphetamines (amphetamine, AP; methamphetamine, MA; methylenedioxy amphetamine, MDA; methylenedioxy methamphetamine, MDMA; methylenedioxy ethylamphetamine, MDEA), ketamine (ketamine, K; norketamine, NK), and opiates (morphine, MOR; codeine, COD; 6-acetylmorphine, 6-AM). This strategy integrated the characteristics of gas chromatography–mass spectrometry (GC–MS) using electron impact ionization (EI) and negative chemical ionization (NCI). Hair samples (25 mg) were washed, cut, and incubated overnight at 25 °C in methanol–trifluoroacetic acid (methanol–TFA). The samples were extracted by solid phase extraction (SPE) procedure, derivatized using heptafluorobutyric acid anhydride (HFBA) at 70 °C for 30 min, and the derivatives analyzed by GC–MS with EI and NCI. The limit of detection (LOD) with GC/EI-MS analysis obtained were 0.03 ng/mg for AP, MA, MDA, MDMA, and MDEA; 0.05 ng/mg for K, NK, MOR, and COD; and 0.08 ng/mg for 6-AM. The LOD of GC/NCI-MS analysis was much lower than GC/EI-MS analysis. The LOD obtained were 30 pg/mg for AP and MDA in GC/EI-MS and 2 pg/mg in GC/NCI-MS. Therefore, the sensitivity of AP and MDA in GC/NCI-MS was improved from 15-fold compared with EI. The sensitivity of AP, MA, MDA, MDMA, MDEA, MOR, and COD was improved from 15- to 60-fold compared with EI. In addition, the sensitivity of 6-AM increased 8-fold through selection of m/z 197 for the quantitative ion. Moreover, K and NK could dramatically improve their sensitivity at 200- and 2000-fold. The integration of GC/EI-MS and GC/NCI-MS can obtain the high sensitivity and complementary results of drugs of abuse in hair. Six hair samples from known drug abusers were examined by this new strategy. These results show that integrating the characteristics of GC/EI-MS and GC/NCI-MS were not only enhancement of the sensitivity but also avoid wrong results and wrong interpretations of correct results.
Keywords: Drugs of abuse; Simultaneous; Hair testing; Multiple ionization;

A hollow-fibre supported liquid membrane (HF-SLM) extraction method has been developed for determination of 11 heterocyclic aromatic amines (HCAs) in human urine samples by using high performance liquid chromatography (HPLC) equipped with an ultraviolet (UV) absorbance detector. These compounds were extracted from an alkaline urine sample (donor phase) into the organic solvent residing in the pores of a polypropylene hollow fibre and then back extracted into an acidic solution (acceptor phase) inside the lumen of the hollow fibre. After extraction, HCAs were analyzed by injecting the analyte enriched acceptor phase into the HPLC. The analyte enrichment factors ranged between 241 and 339 obtained in a 90 min extraction time, and method detection limits (MDL) ranged between 0.1 and 0.5 μg L−1 with relative standard deviation (RSD) values between 3.4% and 11%. The extraction technique employed in this work is easy to use and rapid as it involves only a few minutes manipulation of each sample. It is the most economical sample preparation/preconcentration technique to our knowledge as compared to other microextraction techniques.
Keywords: Heterocyclic aromatic amines; Urine sample; Hollow-fibre supported liquid membrane; High performance liquid chromatography with ultraviolet detector;

A novel method using solid-phase extraction coupled with gas chromatography and flame ionization detector (FID)/electron impact mass spectrometry (EIMS) was developed for the determination of 5α-androst-16-en-3α-ol (androstenol), a steroidal compound belonging to the group of musk odorous 16-androstenes, in truffle fermentation broth. Comparison studies between FID and EIMS indicated two detectors gave similar quantitative results. The highest androstenol concentration of 123.5 ng/mL was detected in Tuber indicum fermentation broth, while no androstenol was found in Tuber aestivum fermentation broth. For the first time, this work confirmed the existence of androstenol in the truffle fermentation broth, which suggested truffle fermentation is a promising alternative for androstenol production on a large scale.
Keywords: 5α-Androst-16-en-3α-ol; Truffle fermentation; Solid-phase extraction; Gas chromatography; Flame ionization detector; Electron impact mass spectrometry;

A novel HPLC-UV/nano-TiO2-chemiluminescence system for the determination of selenocystine and selenomethionine by Yingying Su; He Chen; Ying Gao; Xiaohong Li; Xiandeng Hou; Yi Lv (216-221).
Active oxygen species from the photocatalytic reaction in aqueous solution react with luminol to emit strong chemiluminescence (CL), and this can be inhibited by the UV decomposed-products of selenocystine (SeCys) or selenomethionine (SeMet). Based on this phenomenon, a novel hyphenated technique, HPLC-UV/nano-TiO2-CL, was established for the determination of SeCys and SeMet. The effects of pH, the UV irradiation time, the TiO2 coated on the inner surface of the reaction tubing, and the Co2+ catalyst concentration on the CL intensity and/or chromatographic resolution were systematically investigated. Under these optimized conditions, the inhibited CL intensity has a good linear relationship with the concentration of SeCys in the range of 0.04–10.6 μg mL−1 or SeMet in the range of 0.05–12.4 μg mL−1, with a limit of detection (S/N = 3) of 6.4 μg L−1 for SeCys or 12 μg L−1 for SeMet. As an example, the method was preliminarily applied to the determination of the selenoamino acids in garlic and rabbit serum, with a recovery of 88–104%.
Keywords: Liquid chromatography; Photocatalytic; Chemiluminescence; Selenoamino acids;

Automated GC–MS analysis of free amino acids in biological fluids by Hannelore Kaspar; Katja Dettmer; Wolfram Gronwald; Peter J. Oefner (222-232).
A gas chromatography–mass spectrometry (GC–MS) method was developed for the quantitative analysis of free amino acids as their propyl chloroformate derivatives in biological fluids. Derivatization with propyl chloroformate is carried out directly in the biological samples without prior protein precipitation or solid-phase extraction of the amino acids, thereby allowing automation of the entire procedure, including addition of reagents, extraction and injection into the GC–MS. The total analysis time was 30 min and 30 amino acids could be reliably quantified using 19 stable isotope-labeled amino acids as internal standards. Limits of detection (LOD) and lower limits of quantification (LLOQ) were in the range of 0.03–12 μM and 0.3–30 μM, respectively. The method was validated using a certified amino acid standard and reference plasma, and its applicability to different biological fluids was shown. Intra-day precision for the analysis of human urine, blood plasma, and cell culture medium was 2.0–8.8%, 0.9–8.3%, and 2.0–14.3%, respectively, while the inter-day precision for human urine was 1.5–14.1%.
Keywords: Metabolomics; Amino acids; Gas chromatography–mass spectrometry; Urine; Plasma; Alkyl chloroformate;

A fast and sensitive LC–ESI-MS method is described for the comparative quantification of 16 estrogen metabolites based on the derivatization of estrogens with a novel derivatizing reagent, N-methyl-nicotinic acid N-hydroxysuccinimide ester (C1-NA-NHS). The process introduces a quaternary amine to the analytes, making the analytes permanently charged regardless of the pH of the high-performance liquid chromatography (HPLC) mobile phase. This quaternization resulted in a highly efficient separation of 16 estrogen metabolites in 7 min at a detection level below 1 ng/mL. By using a deuterated derivatizing reagent (C1-d 3-NA-NHS), a complete set of deuterated standards was utilized and used as internal standards in a comparative quantification and recovery study, demonstrating acceptable results over a wide concentration range. A pooled breast cancer serum sample was analyzed using the described method, and 15 estrogens were detected in the range of 80–530 pg/mL.
Keywords: Estrogen; HPLC; Mass spectrometry;

A rapid method to identify and quantify unconjugated progestogens in eggs is presented. Samples were prepared based on matrix solid-phase dispersion (MSPD) using C18 as dispersant, followed by a clean-up step with graphitized carbon black (GCB) solid-phase extraction (SPE) cartridges. The analytes were separated by very high pressure LC on a BEH C18 column for a period of 5 min. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was operated in the positive time-scheduled multi-reaction monitoring mode. Recovery studies were performed at two fortification levels. Average recoveries of the target compounds varied from 83.8% to 111.2% and relative standard deviations ranged from 10.5% to 23.7%. The limits of detection (LODs) and limits of quantitation (LOQs) were in the range of 0.2–2.0 μg kg−1 and 0.6–5.0 μg kg−1, respectively. Investigation of real samples indicated that the range of progesterone in eggs was 9.9–40.0 μg kg−1.
Keywords: Progestogens; Matrix solid-phase dispersion; LC–ESI-MS/MS;

Quantitative determination of cyclic polylactic acid oligomers in serum by direct injection liquid chromatography tandem mass spectrometry by Issey Osaka; Arihumi Yoshimoto; Mikio Watanabe; Masashi Takama; Masahiro Murakami; Hideya Kawasaki; Ryuichi Arakawa (247-250).
Polylactic acid (PLA) is a biodegradable polymer, currently used in pharmaceutical and surgical devices. There is a concern that cyclic polylactic acid (CPLA), which is a by-product of PLA synthesis, may be introduced into the human body as an undesirable contaminant. We carried out a quantitation investigation of the CPLA heptamer (CPLA-7) by liquid chromatography mass spectrometry (LC–MS). We found that CPLA-7 binds strongly with serum proteins and that only 62% of CPLA-7 was recovered after routine deproteination; therefore, we directly injected serum into the LC–MS/MS system after passage through a bovine serum albumin (BSA)-coated chromatographic column and found the recovery of CPLA-7 was improved to 84%, and that the detection (S/N = 3) and quantitation limit (S/N = 10 and below 15% relative standard deviation) were 1.5 and 2.5 ng/mL, respectively. We conclude that direct injection LC–MS/MS, using a BSA column, is a simple and effective quantitative analysis method for CPLA in serum.
Keywords: Cyclic polylactic acid oligomer; LC–MS/MS; Deproteination; Direct injection of serum sample; BSA-coated column;

A sensitive and specific HPLC–MS/MS method was developed for the analysis of mycophenolic acid glucuronide (MPAG) in incubations with human liver microsomes. Incubation samples were processed by protein precipitation with acetonitrile. MPAG and the internal standard phenolphthalein glucuronide were chromatographed on a C18 Synergi Fusion-RP column (100 mm × 2 mm, 4 μm) using gradient elution with a mixture of 1 mM acetic acid in deionized water and 1 mM acetic acid in acetonitrile at a flow rate of 0.22 mL/min. The mass spectrometer was operated with negative electrospray ionization and analysis was carried out in the single reaction monitoring (SRM) mode using the mass transitions of m/z 495 → 319 and m/z 493 → 175 for MPAG and phenolphthalein glucuronide, respectively. The MPAG calibration curve was linear over the concentration range of 1.0–20 μM. The within-day and between-day relative standard deviations ranged from 1.1 to 7.9% and accuracy was within 8%. The simple and reproducible method is suitable for measuring mycophenolic acid glucuronidation in microsomal incubations.
Keywords: Mycophenolic acid; Glucuronidation; Drug metabolism;

Determination of bromide in canine plasma using ion chromatography by S.K. Cox; A.M. Whiton; H.L. Bowman (255-258).
A new ion chromatographic procedure has been developed and validated for the determination of bromide in canine plasma. Following a simple dilution, samples were separated on a Metrosep A Supp 5 column. The mobile phase was an isocratic mixture of 2.2 mM Na2CO3, 1.0 mM NaHCO3, and 1% acetonitrile, with a flow-rate of 0.7 ml/min. The procedure produced a linear curve over the concentration range of 50–2500 μg/ml. The development of the assay permitted the determination of therapeutic levels after oral administration of potassium bromide to dogs being treated for epilepsy.
Keywords: Ion chromatography; Bromide; Epilepsy;