Journal of Chromatography B (v.808, #2)
OFC: Update (OFC).
News Section (N1-N2).
Liquid chromatography method for quantifying N-(4-hydroxyphenyl)retinamide and N-(4-methoxyphenyl)retinamide in tissues by Jitka Vratilova; Tomas Frgala; Barry J Maurer; C Patrick Reynolds (125-130).
A simple and accurate high-performance liquid chromatography (HPLC) method was developed to measure levels of N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and its main metabolite N-(4-methoxyphenyl)retinamide (4-MPR) in tissue. Following ultrasonic extraction of fresh tissue in acetonitrile (ACN), 4-HPR and 4-MPR were measured by HPLC with UV absorbance detection at 340 nm, using isocratic elution with ACN, H2O, and acetic acid. N-(4-ethoxyphenyl)retinamide (4-EPR) was employed as an internal standard. The 4-HPR and 4-MPR recovery in bovine liver or bovine brain tissue samples spiked with known amounts of 4-HPR and 4-MPR ranged from 93 to 110%. The detection limit of the method was 50 ng/ml. The method was tested on actual samples from an athymic (nu/nu) mouse carrying a subcutaneous tumor xenograft originating from SMS-KCNR neuroblastoma cells. The tissues were harvested and analyzed following a 3 day long treatment with intraperitoneal injections of 4-HPR/Diluent-12. 4-HPR and the metabolite 4-MPR were detected and quantitated in the tested tissues including tumor, liver, and brain. This method can be used to quantify 4-HPR and 4-MPR in different tissues to determine the bioavailability of 4-HPR.
Keywords: N-(4-hydroxyphenyl)retinamide; N-(4-methoxyphenyl)retinamide;
Determination of dihydroergocryptine in human plasma and urine samples using on-line sample extraction–column-switching reversed-phase liquid chromatography–mass spectrometry by Gerhard Friedrich; Kurt Appel; Thorsten Rose; Martina Wangemann; Michael Althaus; Klaus Rissler (131-139).
A rapid and sensitive assay for the determination of dihydroergocryptine (DHEC) in human plasma and urine samples with dihydroergotamine (DHET) as the internal standard was developed. The procedure employs on-line sample preparation using an extraction pre-column and an octadecylsilylsilica (ODS) analytical column. After centrifugation human plasma or urine were injected onto the pre-column, concentrated and extracted, back-flushed onto the analytical column and eluted with a binary methanol—aqueous formic acid gradient. Either determination of DHEC as well of its mono- and dihydroxy-metabolites was performed by measurement of the signal responses from MS detection in the selected reaction monitoring (SRM) mode using the transition of the respective parent ions to the common daughter ion at m/z=270.2 amu. The limit of quantitation (LOQ) for determinations of DHEC in both plasma and urine were 25 pg/ml for injected sample volumes of 400 μl. Proportionality of signal responses versus concentration was accomplished within the range of 25–1000 pg/ml. Recovery of target analyte from plasma was 99%. Mean values of the coefficients of variation (CV) for the target analyte in plasma ranged from 1.7 to 13.8% (within-day) and 5.0 to 9.1% (between-day) and accuracy from 91.7 to 102.6% for the within-day and from 95.8 to 98.8% for the between-day measurements. The corresponding values for determinations in urine were 1.7–14.5% (within-day) and 5.3–11.8% (between-day) for CV and 95.8–110.7% (within-day) and 100.1–104.6% (between-day) for accuracy.
Quantification of Doxorubicin and metabolites in rat plasma and small volume tissue samples by liquid chromatography/electrospray tandem mass spectroscopy by Robert D. Arnold; Jeanine E. Slack; Robert M. Straubinger (141-152).
The anthracycline Doxorubicin (DXR) is used widely for the treatment of human malignancies, and drug delivery technologies are under investigation to enhance antitumor selectivity and effectiveness. A liquid chromatography–tandem mass spectroscopy (LC–MS/MS) method was developed to identify and quantify DXR and key metabolites in small-volume biological samples. The assay was linear over the therapeutically relevant concentration range (0.125–10,000 nM); in brain tissue, the lower limit of quantification was 0.247 nM and the sensitivity was 1.4 pg. The ability to quantify DXR and detect metabolite formation may provide insight into the toxicity and bioavailability of drug incorporated into carriers such as liposomes.
Keywords: Doxorubicin; Anthracyclines;
Unified gas chromatographic–mass spectrometric method for quantitating tyrosine metabolites in urine and plasma by Albert L Shroads; George N Henderson; Jang Cheung; Margaret O James; Peter W Stacpoole (153-161).
Tyrosine and many of its catabolites play significant roles in the in the toxicity associated with acquired and congenital forms of hypertyrosinemia. We now report a specific and sensitive GC/MS method for the simultaneous determination of tyrosine metabolites maleylacetone (MA), fumarylacetone (FA), succinylacetone (SA), fumarate and acetoacetate in urine and plasma. Tyrosine metabolites and an internal standard, 2-oxohexanoic acid (OHA), in urine or plasma samples were derivatized to their methyl esters with a 12% boron trifluoride–methanol complex (12%BF3–MeOH). The reaction mixture was extracted with methylene chloride and analyzed by GC/MS, using a selected ion monitoring (SIM) mode. The detection limits were in the range of 0.08–0.4 ng and the quantitation limits were 0.2–2 ng. Most of the intraday and interday coefficients of variation for three concentrations (low, medium and high) of the analytes were below 10%. Sensitivity and selectivity are superior to existing HPLC or enzymatic methods and derivatization of samples is simpler than the traditional silylation of organic acids used for analysis by GC/MS or derivatization to oximes, followed by silylation in the case of the ketoacids, such as SA. Furthermore, the current procedure can be performed in aqueous solution, which results in a high percentage yield without appreciable analyte degradation or formation of side products. Thus far, the method has been successfully applied in the analysis of over 5000 urine and plasma samples from humans and rodents.
Keywords: Tyrosinemia; Tyrosine; Maleylacetone; Fumarylacetone; Succinylacetone; Fumarate; Nitisione;
Liquid chromatographic method for the quantitative determination of N ϵ-carboxymethyllysine in human plasma proteins by Nico C van de Merbel; Cyriel J.A.L Mentink; Gert Hendriks; Bruce H.R Wolffenbuttel (163-168).
The modification of the lysine moieties of proteins to N ϵ-carboxymethyllysine (CML) is supposed to play a major role in the development of long-term complications in patients with diabetes mellitus. This paper presents an analytical method for the quantitative determination of CML in plasma proteins, which could be used for studying the development of diabetic complications. The method is based on isolating proteins from plasma by precipitation with trichloroacetic acid and hydrolysing these under acidic conditions (6 M hydrochloric acid at 110 °C for 20 h) to the individual amino acids. After hydrolysis, CML is derivatised along with the other amino acids to 9-fluorenylmethoxycarbonyl (FMOC) derivatives, which are subsequently separated by reversed-phase column liquid chromatography using a 150 mm × 4.6 mm C8 column and a mobile phase of 25 mM potassium phosphate buffer (pH 2.0) and acetonitrile (80:20 (v/v)) and detected using fluorescence detection (excitation at 260 nm and emission at 310 nm). Quantification of the protein-bound CML content of a plasma sample is achieved using standard addition. The impact of several aspects of the sample preparation and chromatography on method performance is discussed. Method evaluation results are reported and show that this method is capable of determining CML with good accuracy and precision (below 10%) in the relevant concentration range (1–10 μg/ml), with a limit of detection of 0.2 μg/ml.
Keywords: Derivatization, LC; N ϵ-Carboxymethyllysine; Proteins;
On-line coupling of sequential injection with liquid chromatography for the automated derivatization and determination of γ-aminobutyric acid in human biological fluids by Constantinos K. Zacharis; Georgios A. Theodoridis; Anastasios N. Voulgaropoulos (169-175).
The principle of sequential injection analysis (SIA) was exploited to develop a rapid fully automated and efficient pre-column derivatization procedure coupled on-line to liquid chromatography (HPLC). Using the SIA-HPLC derivatization protocol γ-aminobutyric acid (GABA) was determined fluorimetrically in human biological fluids with o-phthaldialdehyde (OPA) as derivatization reagent and minimum sample pretreatment. A lab-built SIA system was used to handle samples, standard solutions and OPA reagent. Appropriate volumes of the reagents were introduced in the holding coil of the SIA system and were mixed on propulsion to the HPLC loop through a suitable reaction coil. The chemical (pH, c(OPA), c(mercaptoethanol)) and instrumental variables (volumes of sample and reagent, reaction time) of the reaction were studied and optimized in terms of maximum sensitivity. The chromatographic variables (gradient composition of the eluent and flow rate) were studied for optimum selectivity and peak characteristics. The developed experimental configuration facilitated fully-automated operation thus minimizing errors in handling. Additionally the method as a whole provided very satisfactory sensitivity, precision and accuracy. Direct determination of GABA in human urine and cerebrospinal fluid (CSF) at μg L−1 (ppb) levels was accomplished, with minimum sample pretreatment.
Keywords: Sequential injection; Derivatization, LC; γ-Aminobutyric acid;
Liquid chromatographic method for determination of four active saponins from Panax notoginseng in rat urine using solid-phase extraction by Lie Li; Jin-Lan Zhang; Yu-Xin Sheng; Guan Ye; Hong-Zhu Guo; De-An Guo (177-183).
Four major active saponins (ginsenosides Rg1, Rb1, Rd and notoginsenoside R1) in Panax notoginseng were determined in rat urine after oral and intravenous administration of total saponins of P. notoginseng (PNS), and the urine samples were treated with solid-phase extraction (SPE) prior to liquid chromatography. A reversed-phase liquid chromatography system with ultraviolet detection and a Zorbax SB-C18 column was used. The within-day and between-day assay coefficients of variation for the four saponins in urine were less than 7% and the recovery of this method was higher than 85%. Using this method, the excretion profile of the drug in rat urine after administration of PNS was revealed for the first time.
Keywords: Panax notoginseng; Saponins; Ginsenosides;
Expressional pattern of known and predicted signaling proteins in seven human cell lines by Daniela Pollak; Kurt Krapfenbauer; Michael Fountoulakis; Andreas Peyrl; Gert Lubec (185-208).
Although a variety of signaling systems and signaling proteins have been described, cell specific expression of these structures has not yet been systematically studied. Human amnion, bronchial epithelial, fibroblast, glial, kidney, lymphocyte and mesothelial cells were subjected to two-dimensional-gel electrophoresis followed by analysis of protein spots by MALDI-TOF and subsequent identification by specific software. A series of well-documented signaling proteins showed cell specific expressional patterns. Five hypothetical proteins—hypothetical 37.5 kDa protein, similar to calsyntenin 1, hypothetical armadillo repeat/plakoglobulin ARM-repeat profile containing protein, 11 days embryo cDNA clone 2700084k13, hypothetical protein flj22171—so far predicted from their nucleic acid sequence only, were identified, complementing already reported signaling cascades. An analytical tool for the concomitant determination of a large series of signaling structures by an antibody independent protein-chemical method is provided.
Keywords: Proteomics; Signaling proteins; Hypothetical proteins;
Gas chromatography-mass spectrometry determination of matrine in human plasma by Dan S.T Sit; Guanghua Gao; Francis C.P Law; Paul C.H Li (209-214).
A method was developed for the quantification of matrine in human plasma using a liquid–liquid extraction procedure followed by gas-chromatography-mass spectrometry (GC/MS) analysis. Deuterated matrine, an internal standard of the analysis, was spiked into the plasma samples before extraction. Linear detection responses were obtained for matrine concentrations ranging from 10 to 500 ng/ml. The intra-day and inter-day precision ranged from 0.4 to 4.0% and 1.0–3.5%, respectively. The intra-day accuracy was between −7.3 and 4.5%. The limit of quantification for matrine was 23 ng/ml. The extraction efficiency averaged about 38%. The validated GC/MS method will be used to quantify matrine in human plasma samples collected in a clinical trial study.
Rapid and sensitive liquid chromatography–tandem mass spectrometric method for the quantitation of metformin in human plasma by Yingwu Wang; Yunbiao Tang; Jingkai Gu; J.Paul Fawcett; Xu Bai (215-219).
A rapid and sensitive liquid chromatography–tandem mass spectrometric (LC–MS–MS) method for the determination of metformin in human plasma using phenformin as internal standard has been developed and validated. Sample preparation of plasma involved acidification with acetic acid, deproteination with acetonitrile and washing with dichloromethane. Samples were then analyzed by HPLC on a short Nucleosil C18 column (5 μm, 50 mm × 4.6 mm i.d.) using a mobile phase consisting of acetonitrile:methanol:10 mM ammonium acetate pH 7.0 (20:20:60, v/v/v) delivered at 0.65 ml/min. Detection was performed using an Applied Biosystems Sciex API 4000 mass spectrometer set at unit resolution in the multiple reaction monitoring (MRM) mode. Atmospheric pressure chemical ionization (APCI) was used for ion production. The assay was linear over the range 1–2000 ng/ml with intra- and inter-day precision of <8.6% and accuracy in the range 91–110%. The limit of detection was 250 pg/ml in plasma. The method was successfully applied to a clinical pharmacokinetic study of an extended-release tablet of metformin hydrochloride (500 mg) administered as a single oral dose.
Determination of flumazenil in human plasma by liquid chromatography–electrospray ionisation tandem mass spectrometry by M Lavén; L Appel; R Moulder; N Tyrefors; K Markides; B Långström (221-227).
A liquid chromatography–electrospray ionisation–tandem mass spectrometry (LC–ESI–MS/MS) method was developed to determine unlabelled flumazenil (Ro 15-1788) in human plasma in [11C]flumazenil positron emission tomography (PET) studies. N-Methyl tri-deuterated flumazenil was used as an internal standard. The analyte and internal standard were extracted from plasma samples using solid-phase extraction, with a recovery of 78%. This was determined through the convenience of radioactivity measurements of 11C-labelled flumazenil. The evaporated and reconstituted eluate was analysed by LC–ESI–MS/MS. The calibration curve was linear over the tested concentration range of 0.05–0.5 nM (15–150 pg/ml) with a correlation coefficient, R 2, of 0.998±0.001. A high precision was achieved, with mean intra-assay and inter-assay relative standard deviations of at most 6 and 7%, respectively. The accuracy of the method ranged from 95 to 104%. As a proof of concept, the validated method was applied in the determination of flumazenil in plasma from two healthy volunteers participating in a PET study with three repeated investigations. A bolus-infusion protocol was used to achieve a constant concentration level of flumazenil. The average plasma concentrations ranged from 0.11 and 0.19 nM and all measurements were within the calibration standard range. The flumazenil concentrations were relatively constant within each scan and the average intra-scan precision was 15%.
An estimation of the exposure to organophosphorus pesticides through the simultaneous determination of their main metabolites in urine by liquid chromatography–tandem mass spectrometry by F Hernández; J.V Sancho; O.J Pozo (229-239).
A rapid method has been developed for the determination of 4-nitrophenol (PNP) (parathion and methyl-parathion metabolite) and 3-methyl-4-nitrophenol (3-Me-PNP) (fenitrothion metabolite) in human urine by coupled-column liquid chromatography combined with tandem mass spectrometry (LC–LC–MS/MS). The LC–LC–MS/MS approach allows the determination at sub-ppb level of free metabolites by injecting the urine directly into the system and the total metabolites after a simple enzymatic hydrolysis. The method has been validated, obtaining limits of detection of 0.1 and 0.2 μg/L for 4-nitrophenol and 3-methyl-4-nitrophenol, respectively. Additionally, a multi-residue LC–MS/MS method is proposed in order to evaluate the levels of other parathion and methyl parathion metabolites. This approach allows the simultaneous determination of dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP), 4-nitrophenolsulphate and 4-nitrophenolglucuronide without tedious sample treatments. The applicability of both methods is demonstrated by applying them to various urine samples from an unexposed population and a grower who applied methyl parathion. The combination of both methods allows a general overview on the presence of different metabolites (free and conjugated) and the concentration ratios between them, giving useful information on organophosphorus pesticides metabolism and excretion.
Keywords: Organophosphorus pesticides; 4-Nitrophenol; 3-Methyl-4-nitrophenol;
Determination of single-cell gene expression in Arabidopsis by capillary electrophoresis with laser induced fluorescence detection by Xin Liu; Li Ma; Jian-Feng Zhang; Ying-Tang Lu (241-247).
We report a new method for the measurement of gene expression in single cells of Arabidopsis using capillary electrophoresis with laser-induced fluorescence (CE–LIF) detection. Initially, the quantitative analysis of APETALA2 (AP2) and LEAFY (LFY) was performed by CE–LIF method. The detection limits of AP2 and LFY can reach 0.08 and 0.04 ng/ml (signal-to-noise ratio = 3), respectively. This protocol coupling with single-cell reverse transcriptase-polymerase chain reaction (SC-RT-PCR) has been used to monitor LFY and AP2 expression in individual cells from the shoot apical meristem, leaf, root, and stem of Arabidopsis, simultaneously. The effect of PCR cycle number on PCR product concentrations has been discussed. The changes of LFY expression were determined at single-cell level in different Arabidopsis tissues. The relationship between gibberallic acid (GA) and LFY expression was also revealed by this method. It was shown that the combination between CE–LIF and SC-RT-PCR could provide a highly sensitive and selective tool for the determination of different gene expression at single-cell level in specific tiny plant tissues.
Keywords: Arabidopsis; Gene expression; Single-cell transcriptase–polymerase chain reaction;
Characterization of spirochetal isolates from arthropods collected in South Moravia, Czech Republic, using fatty acid methyl esters analysis by Leona Čechová; Eva Durnová; Silvie Šikutová; Jiřı́ Halouzka; Miroslav Němec (249-254).
Aim of this study was to evaluate cellular fatty acid analysis for characterization of spirochetes. Strains were isolated from arthropods collected in South Moravia, Czech Republic. Fatty acid methyl esters (FAME) profile was determined for five Borrelia burgdorferi sensu lato (s.l.) strains isolated from Ixodes ricinus ticks, one “Spironema culicis” strain recovered from mosquito Culex pipiens and seven spirochetal strains (not identified yet) isolated from mosquitoes and blackflies. Analysis was performed using a gas chromatography column in conjunction with Microbial Identification System Sherlock (MIDI Inc., Newark, DE, USA). Results obtained on the basis of cluster analysis of FAME profiles showed, that the B. burgdorferi sensu lato isolates could be well separated from other spirochetal isolates. We recommended method used in this study as a useful tool for preliminary identification of spirochetes isolated from ticks and dipterans.
Keywords: Spirochetes; Arthropods; Fatty acid methyl esters;
SdFFF monitoring of cellular apoptosis induction by diosgenin and different inducers in the human 1547 osteosarcoma cell line by C Corbière; S Battu; B Liagre; P.J.P Cardot; J.L Beneytout (255-262).
Apoptosis is one of the most important phenomena of cellular biology. Sedimentation field flow fractionation (SdFFF) has been described as an effective tool for cell separation, respecting integrity and viability. Because SdFFF takes advantage of intrinsic properties of eluted cells (size, density, shape or rigidity), we investigated the capacity of SdFFF in monitoring the early and specific biophysical modifications which occurred during cellular apoptosis induction. Then, we used, as an in vitro cellular apoptosis model, the association between human 1547 osteosarcoma cells and diosgenin, a plant steroid known to induce apoptosis. Four other molecules were studied: hecogenin, tigogenin, staurosporine and MG132. Our results demonstrated a correlation between SdFFF elution profile changes (peak shape modification and retention ratio evolution) and effective apoptosis induction. For the first time, we demonstrated that SdFFF could be used to monitor apoptosis induction as early as 6 h incubation, suggesting different applications such as screening series of molecules to evaluate their ability to induce apoptosis, or sorting apoptotic cells to study apoptosis pathway.
Keywords: Sedimentation field flow fractionation; Apoptosis; Plant steroids; Diosgenin; Staurosporine; MG132;
Determination of long-chain fatty acid acyl-coenzyme A compounds using liquid chromatography–electrospray ionization tandem mass spectrometry by T Mauriala; K.H Herzig; M Heinonen; J Idziak; S Auriola (263-268).
Acyl-CoAs have important role in fat and glucose metabolism of the cells. In this study we have developed an on-line HPLC–ESI-MS/MS method for determination of long-chain acyl-CoA compounds in rat liver samples. Six long-chain acyl-CoAs (C16:0, C16:1, C18:0, C18:1, C20:0 and C20:4) were separated with a C4 reversed-phase column using triethylamine acetate and acetonitrile gradient. Negative electrospray ionization is very suitable for acyl-CoA compounds and excellent MS/MS spectra for long-chain acyl-CoAs can be obtained. MS/MS method with an ion trap mass spectrometer makes it possible to identify and quantitate individual acyl-CoAs simultaneously. The method proved to be sensitive enough for determination of all compounds of interest using 0.4–0.7 g of tissue and was validated in the range of 0.1–15.0 pmol/μl.
Keywords: Long-chain acyl-coenzyme A compounds;
Investigation of volatile biomarkers in lung cancer blood using solid-phase microextraction and capillary gas chromatography–mass spectrometry by Chunhui Deng; Xiangmin Zhang; Ning Li (269-277).
In the present work, solid-phase microextraction (SPME) and gas chromatography–mass spectrometry (GC–MS) was developed for investigation of lung cancer volatile biomarkers. Headspace SPME conditions (fiber coating, extraction temperature and extraction time) and desorption conditions were optimized and applied to determination of volatiles in human blood. To find the biomarkers of lung cancer, investigation of volatile compounds in lung cancer blood and control was performed by using the present method. Concentrations of hexanal and heptanal in lung cancer blood were found to be much higher than those in control blood. The two molecules of hexanal and heptanal were regarded as biomarkers of lung cancer. By comparison of volatiles in breath and in blood, it is demonstrated that hexanal and heptanal in breath were originated from blood and screening of lung cancer by breath analysis be feasible. These results show that SPME/GC–MS is a simple, rapid and sensitive method very suitable for investigation of volatile disease markers in human blood.
Keywords: Volatile biomarkers; Hexanal; Heptanal;
Proteomic analysis of an orthotopic neuroblastoma xenograft animal model by Natascia Campostrini; Jennifer Pascali; Mahmoud Hamdan; Hubert Astner; Danilo Marimpietri; Fabio Pastorino; Mirco Ponzoni; Pier Giorgio Righetti (279-286).
Neuroblastoma is the most common extracranial solid tumour of childhood and comprises up to 50% of malignancies among infants. There is a great need of designing novel therapeutic strategies and proteome analysis is one approach for defining markers useful for tumour diagnosis, as well as molecular targets for novel experimental therapies. We started by comparing healthy adrenal glands (which are the election organs developing primary neuroblastoma, NB, tumours) and adrenal glands carrying primary NB tumours, taken from nude mice. Standard maps of healthy and tumour samples were generated by analysis with the PDQuest software. The comparison between such maps showed up- and down-regulation of 84 polypeptide chains, out of a total of 700 spots detected by a fluorescent stain, Sypro Ruby. Spots that were differentially expressed between the two groups, were analysed by MALDI-TOF mass spectrometry and 14 of these spots were identified so far. Among these proteins, of particular interest are the down-regulated proteins adrenodoxin (21-folds), carbonic anhydrase III (eight-folds) and aldose reductase related protein I (eight-folds), as well as the up-regulated protein peptidyl-propyl cis–trans isomerase A (five-folds). Moreover new proteins, which were absent in control samples, were expressed in tumour samples, such as nucleophosmin (NPM) and stathmin (oncoprotein 18).
Keywords: Proteomics; Neuroblastoma; Two-dimensional maps;
Detection of endogenous boldenone in the entire male horses by Emmie N.M. Ho; Kenneth C.H. Yiu; Francis P.W. Tang; Louis Dehennin; Philippe Plou; Yves Bonnaire; Terence S.M. Wan (287-294).
Boldenone (1,2-dehydrotestosterone) is a common veterinary anabolic agent. Its structure is very similar to testosterone. Testosterone is endogenous in the horse, whereas there has been no report concerning the detection of endogenous boldenone. This paper reports the direct observation of sulphate conjugate of boldenone in equine urine from entires. The detection procedures involved solid-phase extraction, immunoaffinity column (IAC) purification, and then LC–MS–MS analysis on a Q-ToF instrument. The identification of boldenone sulphate has provided direct evidence for the endogenous nature of boldenone in entire male horses. Quantification data for the normal level of boldenone in Hong Kong racehorses will also be discussed.
Subcellular metabolite profiles of the parent CCRF-CEM and the derived CEM/C2 cell lines after treatment with doxorubicin by Adrian B Anderson; Edgar A Arriaga (295-302).
Micellar electrokinetic capillary chromatography with laser-induced fluorescence detection was used to detect the differences in doxorubicin metabolite accumulation in four subcellular fractions isolated from the CCRF-CEM and the CEM/C2 human leukemia cell lines. Five fluorescent metabolites and doxorubicin make up the metabolite profile of these cell lines upon treatment with 10 μM doxorubicin for 12 h, cell lysis, and fractionation by differential centrifugation. Based on the relative electrophoretic mobility of synthetic standards, we tentatively identify one metabolite as 7-deoxydoxorubicinone and suggest that doxorubicinone is not among those metabolites detected. Although the obvious difference between the derived cell line (CEM/C2) and the parent cell line (CCRF-CEM) is the decreased topoisomerase I activity in the former, the results presented here indicate that each cell line has a unique distribution of metabolites in each one of four subcellular fractions: nuclear-enriched, heavy-organelle-enriched, light-organelle-enriched, and cytoplasmic fractions.
Keywords: Metabolism; Micellar electrokinetic capillary chromatography; Doxorubicin;
Author Index to Vol. 808 (303-305).
Compound Index to Vol. 808 (307-309).