Journal of Chromatography B (v.877, #13)

Quantitative analysis of biomarkers by LC–MS/MS by Jeffrey Cummings; Richard Unwin; Timothy D. Veenstra (1221).

The search for disease markers is not new; however, with the emergence of new technologies such as nano-HPLC and electrospray ionization and time of flight mass spectrometry, the search has intensified considerably. Genomic, proteomic and metabolomic technologies are being used to search for novel disease markers. In this manuscript emphasis will be on different HPLC and MS methods that are used to search for metabolites and proteins that can be used for the discovery of novel, sensitive and specific disease biomarkers. Definitions of terms such as sensitivity, specificity, and protein profiles will be given. Methods used for effective fractionation, separation and quantitation of proteins and peptides using HPLC/MS will be discussed and examples are presented. A brief discussion of electrophoretic procedures used for protein fractionation and biomarker discovery is also included.
Keywords: Disease biomarkers; HPLC–MS/MS; Proteomics; Metabolomics;

Multiple reaction monitoring for quantitative biomarker analysis in proteomics and metabolomics by Neil R. Kitteringham; Rosalind E. Jenkins; Catherine S. Lane; Victoria L. Elliott; B. Kevin Park (1229-1239).
The conventional pipeline for biomarker development involves a discovery phase, typically conducted by mass spectrometry (MS), followed by validation and clinical application, usually on an alternative platform, such as immunoassay. Whilst this approach is suitable for the development of single biomarkers, with the current drive towards larger panels of multiplexed biomarkers, the process becomes inefficient and costly. Consequently, the emphasis is now shifting towards performing full biomarker discovery, qualification and quantification on the same technology platform. The ease of multiplexing and ability to determine protein modifications makes MS an attractive alternative to antibody-based technologies. In addition, developments in quantitative MS, through the application of stable isotope labelling and scanning techniques, such as multiple reaction monitoring (MRM), have greatly enhanced both the specificity and sensitivity of MS-based assays to the point that they can rival immunoassay for some analytes. This review focuses on the application of MRM for quantitative MS analysis, particularly with respect to proteins and peptides.
Keywords: Multiple reaction monitoring; Biomarker; Proteomics; Metabolomics; Metabonomics; Mass spectrometry; Quantitative; Triple quadrupole;

The potential for development of personalised medicine through the characterisation of novel biomarkers is an exciting prospect for improved patient care. Recent advances in mass spectrometric (MS) techniques, liquid phase analyte separation and bioinformatic tools for high throughput now mean that this goal may soon become a reality. However, there are challenges to be overcome for the identification and validation of robust biomarkers. Bio-fluids such as plasma and serum are a rich source of protein, many of which may reflect disease status, and due to the ease of sampling and handling, novel blood borne biomarkers are very much sought after. MS-based methods for high throughput protein identification and quantification are now available such that the issues arising from the huge dynamic range of proteins present in plasma may be overcome, allowing deep mining of the blood proteome to reveal novel biomarker signatures for clinical use. In addition, the development of sensitive MS-based methods for biomarker validation may bypass the bottleneck created by the need for generation and usage of reliable antibodies prior to large scale screening. In this review, we discuss the MS-based methods that are available for clinical proteomic analysis and highlight the progress made and future challenges faced in this cutting edge area of research.
Keywords: Biomarker discovery; Mass spectrometry; Quantitative proteomics;

The search for biomarkers in biological fluids that can be used for disease diagnosis and prognosis using mass spectrometry has emerged to become a state-of-the-art methodology for clinical proteomics. Poor cross platform comparison of the findings, however, makes the need for comparison studies probably as urgent as the need for new ones. It is now increasingly recognized that standardized statistical and bioinformatics approaches during data processing are of utmost importance for such comparisons. This paper reviews two of the currently most promising methods, namely LC–MS and CE-MS techniques, and software tools used to analyze the huge amount of data they generate. We further review the statistical issues of feature selection and sample classification.
Keywords: LC–MS; CE-MS; Biomarkers; Clinical proteomics; Statistical data analysis;

Protein biomarkers are used for various purposes in drug development and clinical diagnosis and prognosis. In this review, a fit-for-purpose method validation approach is discussed that fulfills the needs of exploratory and advanced applications in both the pharmaceutical and diagnostic arenas. Method validation for protein biomarkers is typically applied to ligand binding assays (LBA) although hyphenated mass spectrometric methods can be used as adjunct methodologies to confirm LBA specificity or provide valuable information during early discovery or demonstrative phases of a novel biomarker. Pre-analytic variables of protein biomarkers, such as the purpose of the intended application, analyte(s), biological matrix, availability of reference standard, calibrator matrix, assay platform, and sample collection/handling, must be considered in any method development and validation plan. Method validation for exploratory applications involves basic experiments for assay range finding, accuracy and precision, selectivity, specificity, and minimal stability. For advanced method validation, more rigorous tests with a wider scope are performed. These tests include additional patient population ranges, more runs on accuracy and precision from multiple analysts/reagent lots/instruments, selectivity and specificity tests using patient samples, and stability tests subjected to conceivable conditions over long-term use. Differences in biomarker method validation for drug development vs. clinical diagnosis and issues of using developmental commercial kits are discussed. The co-development of biomarkers for drug development and diagnostics presents collaborative opportunities between the pharmaceutical and diagnostic sectors.
Keywords: Method validation; Protein biomarkers; Drug development; Clinical diagnosis/prognosis; Ligand binding assay; Mass spectrometric method;

Leukotriene (LT) B4 is a potent inflammatory lipid mediator that has been involved in the pathophysiology of respiratory diseases including asthma. Exhaled breath condensate (EBC) is a non-invasive method to sample secretions from the airways. LC/MS/MS techniques for measuring LTB4 concentrations in EBC have been developed and are suitable for an accurate quantitative assessment of its concentrations in EBC. LC/MS/MS for other eicosanoids including 8-isoprostane, a marker of oxidative stress, and cysteinyl-LTs have been developed. This article, mainly focused on LTB4, presents the analytical aspects of the LC/MS/MS techniques for measuring LTB4 and 8-isoprostane in EBC, provides examples of their application to the assessment of airway inflammation in patients with asthma and other respiratory diseases, and discusses their potential utility for non-invasive monitoring of drug therapy.
Keywords: Leukotriene B4; Isoprostanes; Exhaled breath condensate; Liquid chromatography/mass spectrometry; Asthma; Non-invasive monitoring; Airway inflammation;

Influence of clotting time on the protein composition of serum samples based on LC–MS data by Natalia I. Govorukhina; Marcel de Vries; Theo H. Reijmers; Péter Horvatovich; Ate G.J. van der Zee; Rainer Bischoff (1281-1291).
Many large, disease-related biobanks of serum samples have been established prior to the widespread use of proteomics in biomarker research. These biobanks may contain relevant information about the disease process, response to therapy or patient classifications especially with respect to long-term follow-up that is otherwise very difficult to obtain based on newly initiated studies, particularly in the case of slowly developing diseases. An important parameter that may influence the composition of serum but that is often not exactly known is clotting time. We therefore investigated the influence of clotting time on the protein and peptide composition of serum by label-free and stable-isotope labeling techniques. The label-free analysis of trypsin-digested serum showed that the overall pattern of LC–MS data is not affected by clotting times varying from 2 to 8 h. However, univariate and multivariate statistical analyses revealed that proteins that are directly involved in blood clot formation, such as the clotting-derived fibrinopeptides, change significantly. This is most easily detected in the supernatant of acid-precipitated, immunodepleted serum. Stable-isotope labeling techniques show that truncated or phosphorylated forms of fibrinopeptides A and B increase or decrease depending on clotting time. These patterns can be easily recognized and should be taken into consideration when analyzing LC–MS data using serum sample collections of which the clotting time is not known. Next to the fibrinopeptides, leucine-rich alpha-2-glycoprotein (P02750) was shown to be consistently decreased in samples with clotting times of more than 1 h. For prospective studies, we recommend to let blood clot for at least 2 h at room temperature using glass tubes with a separation gel and micronized silica to accelerate blood clotting.
Keywords: Biomarker; Clotting time; LC–MS; Proteomics; Serum;

Determination of malondialdehyde in human plasma by fully automated solid phase analytical derivatization by Heather L. Lord; Jack Rosenfeld; Vitaly Volovich; Dinesh Kumbhare; Bill Parkinson (1292-1298).
Analytical derivatization (AD) increases the sensitivity of analysis by one to three orders of magnitude, stabilizes labile analytes and converts them into readily extractable products. Using a variant of this technique, we applied solid phase analytical derivatization (SPAD) to fully automate extraction, derivatization and liquid chromatography. The resulting device (AutoSPAD) determined malonyldialdehyde (MDA) from biological fluids. This biomarker of oxidative stress is highly water-soluble (500 g/L at pH 7), chemically labile and lacks any functionality that enables detection at high sensitivity. AutoSPAD utilizes column-switching technology to load DANSYL hydrazine onto the solid phase, pass the biological sample over the resulting reactor bed for derivatization on the surface to form a hydrophobic derivative suitable for increasing sensitivity of any other LC technique including LC–MS/MS. The hydrophobic solid phase retains the derivative during washing steps, following which AutoSPAD transfers the derivatized extract to the analytical column for separation and detection by fluorescence. In plasma, however, MDA exists both in free form and covalently bound to protein. Measuring MDA from plasma, therefore, required identification of appropriate protein precipitation and hydrolysis conditions. Under these conditions, the DANSYL derivative formed at only one aldehydic position but did not cyclize as reported for other reactions between hydrazine reagents and MDA. The calibration curve using ∼7 μL of plasma was linear (r 2  = 0.999) in the physiological range (0.1–3 μg/mL) and the relative standard deviation of replicate determinations at 1 μg/mL was less than 5%.
Keywords: Solid phase analytical derivatization; Analytical derivatization; Malondialdehyde; Carbonyls; DANSYL hydrazine; High performance liquid chromatography; Fluorescence; Oxidative stress; Biomarkers; Mass spectrometry;

Real-time evaluation of experimental variation in large-scale LC–MS/MS-based quantitative proteomics of complex samples by Yishai Levin; Lan Wang; Erin Ingudomnukul; Emanuel Schwarz; Simon Baron-Cohen; András Palotás; Sabine Bahn (1299-1305).
Quantitative proteomic profiling is becoming a widely used approach in systems biology and biomarker discovery. There is a growing realization that quantitative studies require high numbers of unpooled samples for increased statistical power. Large-scale quantitative analyses require an added degree of stringency due to the lengthy study periods and reliance on stability of analytical instrumentation. We present the inclusion of quality control samples alongside clinical samples in the preparation and nanoLC–MS analysis pipelines. These serve the purpose of monitoring, evaluating and reporting experimental variation measured in real-time. This concept is shown for two types of complex biological samples: serum samples and fibroblast samples. In both studies QC samples were added among dozens of clinical ones and analyzed using a label-free quantitative proteomic platform.
Keywords: Proteomics; LC–MS; MSE; NanoUPLC; Serum; Fibroblasts; QC; Serum; Biomarker discovery;

Robustness and accuracy of high speed LC–MS separations for global peptide quantitation and biomarker discovery by Johan Lengqvist; Jorge Andrade; Yang Yang; Gunvor Alvelius; Rolf Lewensohn; Janne Lehtiö (1306-1316).
The present work investigates qualitative and quantitative variability for rapid liquid chromatography–mass spectrometry (LC–MS) analyses of tryptic digests of total mammalian cell lysate. Experimental variability is characterized in a global manner across technical replicates using label-free quantification software (DeCyder MS 2.0). The effects of a novel time-alignment algorithm are described. Although effective to correct for retention time shifts the time-alignment tool adds only minute benefits for ultra performance (UP)LC–MS data. In differential display experiments, quantitative changes down to 3.5% of the experimental dynamic range could be accurately detected (p  < 0.001). In 17 min analyses, almost 8500 peptide features were detected following injection of ∼1 μg of total cytoplasmic digest. Ion intensity coefficient of variance were <15% for the majority (89%) of all detected peaks. Carry-over of double-charged (tryptic peptide) species was very low (<0.26%). Although the number of peptides detected was highly consistent across replicates, only 58% could be matched to all runs (n  = 5). However, 90% matched to ≥3/5 runs, indicating the importance of replicate runs. The method presented allows high sample throughput which is essential for clinical biomarker discovery. The results are highly encouraging, especially in light of the dynamic range improvements that are presently becoming available on quadrupole time-of-flight instruments.
Keywords: Peptide separation; Biomarker discovery; Quantitative accuracy; Retention time alignment;

The use of proteome similarity for the qualitative and quantitative profiling of reperfused myocardium by Johannes P.C. Vissers; Sandrine Pons; Anne Hulin; Renaud Tissier; Alain Berdeaux; Joanne B. Connolly; James I. Langridge; Scott J. Geromanos; Bijan Ghaleh (1317-1326).
An LC–MS-based approach is presented for the identification and quantification of proteins from unsequenced organisms. The method relies on the preservation of homology across species and the similarity in detection characteristics of proteomes in general. Species related proteomes share similarity that progresses from the amino acid frequency distribution to the complete amino sequence of matured proteins. Moreover, the comparative analysis between theoretical and experimental proteome distributions can be used as a measure for the correctness of detection and identification obtained through LC–MS-based schemes. Presented are means to the identification and quantification of rabbit myocardium proteins, immediately after inducing cardiac arrest, using a data-independent LC–MS acquisition strategy. The employed method of acquisition affords accurate mass information on both the precursor and associated product ions, whilst preserving and recording the intensities of the ions. The latter facilitates label-free quantification. The experimental ion density observations obtained for the rabbit sub proteome were found to share great similarity with five other mammalian samples, including human heart, human breast tissue, human plasma, rat liver and a mouse cell line. Redundant, species-homologues peptide identifications from other mammalian organisms were used for initial protein identification, which were complemented with peptide identifications of translated gene sequences. The feasibility and accuracy of label-free quantification of the identified peptides and proteins utilizing above mentioned strategy is demonstrated for selected cardiac rabbit proteins.
Keywords: Proteome similarity; Ion density; LC–MS; Data-independent scanning; Label-free quantitative proteomics; Reperfused myocardium;

Increased levels of estrogen metabolites (EM) are associated with cancers of the reproductive system. One potential dietary source of EM is milk. In this study, the absolute quantities of unconjugated (free) and unconjugated plus conjugated (total) EM were measured in a variety of commercial milks (whole, 2%, skim, and buttermilk). The results show that the milk products tested contain considerable levels of EM; however, the levels of unconjugated EM in skim milk were substantially lower than that observed in whole milk, 2% milk, and buttermilk. Whole milk contained the lowest overall levels of EM while buttermilk contained the highest. As anticipated, soy milk did not contain the mammalian EM measured using this method. The relatively high levels of catechol estrogens detected in milk products support the theory that milk consumption is a source of EM and their ingestion may have a dietary influence on cancer risk.
Keywords: Estrogen metabolites; Milk; Breast cancer; Biomarkers; Selected reaction monitoring mass spectrometry;

Quantitative proteomic analysis of follicular lymphoma cells in response to rituximab by Kathryn L. Everton; David R. Abbott; David K. Crockett; Kojo S.J. Elenitoba-Johnson; Megan S. Lim (1335-1343).
Rituximab is a monoclonal antibody that targets the uniquely expressed B-cell CD20 receptor. Although recently approved for treatment of follicular lymphomas, the intracellular events that occur when rituximab binds to CD20 are largely unknown. Quantitative proteomic analysis of B-cell lymphoma-derived cells exposed to rituximab was performed. Differentially expressed proteins belonged to functional groups involved in migration, adhesion, calcium-induced signaling, ubiquitination, and components of the phosphoinositol and NF-κB pathways. Our studies reveal the proteomic consequences of rituximab treatment and provide novel insights into the mechanism of action of the drug in susceptible B-cell lymphoproliferative disorders.
Keywords: Rituximab; Drug therapy; Protein interactions; MS/MS; In silico;

Proteomic approaches are used to identify biomarkers, to monitor pathological changes inside of cells and for a better diseases diagnosis. Comparable changes in protein homeostasis also occur in differentiating cells and proteomic techniques should be suitable to identify biomarkers that indicate different steps of cellular development. The C3 exoenzyme from Clostridium botulinum (C3bot) inactivates Rho GTPases and induces morphological cellular changes like cell rounding and neurite outgrowth [G. Ahnert-Hilger, M. Höltje, G. Grosse, G. Pickert, C. Mucke, B. Nixdorf-Bergweiler, P. Boquet, F. Hofmann, I. Just, J. Neurochem. 90 (2004) 9]. To investigate these observations further a comparative proteomic approach has been chosen to elucidate C3bot effects in the neuroblastoma cell line model SH-SY5Y. The screening method applied for biomarker detection was based on the stable isotope approach isobaric tagging for relative and absolute quantification (iTRAQ). Proteins of C3bot-treated and untreated cells were digested and peptides were labeled by the iTRAQ reagent, combined, and separated by means of a two-dimensional nano-HPLC system. Peptide analysis was performed in a MALDI-TOF/TOF mass spectrometer. Identification and quantification of peptides and their corresponding proteins were accomplished by MS/MS spectra analysis. Overall, five replicate measurements identified 355 different proteins of which 235 were accessible for quantification. C3bot altered the concentration of 55 proteins (at least 1.3-fold) and several proteins were identified as possible biomarker candidates that indicate C3bot-induced cellular changes.
Keywords: iTRAQ; Multidimensional chromatography; LC-MALDI; Neuronal cells; SHSY-5Y cells;

The reproducibility of a metabolomics method has been assessed to identify changes in tumour cell metabolites. Tissue culture media extracts were analyzed by reverse phase chromatography on a Waters Acquity T3 column with a 13 min 0.1% formic acid: acetonitrile gradient on Agilent and Waters LC-Q-TOF instruments. Features (m/z, RT) were extracted by MarkerLynx™ (Waters) and Molecular Feature Extractor (Agilent) in positive and negative ionization modes. The number of features were similar on both instruments and the reproducibility of ten replicates was <35% signal variability for ∼50% and 40% of all ions detected in positive and negative ionization modes, respectively. External standards spiked to the matrix showed CVs <25% in peak areas within and between days. U87MG glioblastoma cells exposed to the PI 3-Kinase inhibitor LY294002 showed significant alterations of several confirmed features. These included glycerophosphocholine, already shown by NMR to be modulated by LY294002, highlighting the power of this technology for biomarker discovery.
Keywords: Metabolomics; Reproducibility; Glioblastoma cells; Q-TOF; PI 3-Kinase inhibitor; Validation;

Absolute ProGRP quantification in a clinical relevant concentration range using LC–MS/MS and a comprehensive internal standard by Bjørn Winther; Petter Moi; Marianne S. Nordlund; Niclas Lunder; Elisabeth Paus; J. Léon E. Reubsaet (1359-1365).
The objective of this study was to develop a method using liquid chromatography with tandem mass spectrometric detection for the absolute quantification of the small cell lung cancer biomarker ProGRP in human serum, using its tryptic signature peptide NLLGLIEAK. The samples were precipitated for most of its proteins using acetonitrile prior to tryptic digestion. Further sample clean-up and enrichment was achieved by the use of an on-line restricted access media column, followed by separation on a BioBasic C8 column. Detection and quantification was carried out by operating a triple quadrupole MS in the selected reaction monitoring mode. This setup allowed analysis of realistic samples and detections limits in human serum of 150 pg ProGRP on column. Using an internal standard derived from the parent ProGRP after acetylation of the lysine side chain allowed better quantification through variation correction in all sample pretreatment steps, trypsination included.
Keywords: Biomarker; Small cell lung cancer; ProGRP; Tryptic peptide; Signature peptide; LC–MS/MS; Absolute quantification; MRM; Internal standard; NHS-acetylation;

Recently, it was reported that oxidized phosphatidylcholine shows biological activities via scavenger receptor CD36 or Toll-like receptor 4 (TLR4)-TRIF. Thus, the analysis of oxidized phospholipids is essential in understanding these biological roles. Here, we report an analytical method for oxidized phosphatidylcholines using multiple reaction monitoring (MRM) with theoretically expanded data sets. This analytical method was performed by a quadrupole linear ion trap mass spectrometer with ultra performance LC (UPLC). To investigate whether this established analytical method was applicable to biological samples, we performed variation analysis of oxidized PCs using a myocardial ischemia-reperfusion model. Most oxidized PCs were detected in higher amounts in the ischemic myocardium than in the non-ischemic myocardium. From these application results, this established method is a valuable tool for the global analysis of oxidized PCs. In the future, our study can provide further understanding of how oxidized phospholipids are produced and are correlated to various diseases.
Keywords: Oxidized phosphatidylcholine; Multiple reaction monitoring; Liquid chromatography; Mass spectrometry;

Application of a stable-isotope dilution technique to study the pharmacokinetics of human 15N-labelled S-nitrosoalbumin in the rat: Possible mechanistic and biological implications by Athanasia Warnecke; Piet Luessen; Jörg Sandmann; Milos Ikic; Stefan Rossa; Frank-Mathias Gutzki; Dirk O. Stichtenoth; Dimitrios Tsikas (1375-1387).
In the year 1992, S-nitrosoalbumin (SNALB) has been proposed to be the most abundant physiological carrier and pool of nitric oxide (NO) activity in human circulation, by which NO-dependent biological functions are regulated. The concentration, the metabolism and the mechanisms of the biological actions of SNALB are controversial and still incompletely understood. Moreover, the suitability of SNALB as a biomarker of diseases associated with altered NO bioactivity in human circulation has not been demonstrated convincingly so far. In the present study, we report on the development and application of a stable-isotope technique to study the pharmacokinetics of 15N-labelled SNALB (S15NALB) in anesthetized rats. S15NALB was synthesized from albumin isolated by affinity chromatography from freshly prepared human plasma. This technique was also applied to study and quantify the formation of S15NALB from endogenous rat plasma albumin and intravenously applied S-[15N]nitrosoglutathione (GS15NO) or S-[15N]nitrosocysteine (S15NC) in anesthetized rats. In these investigations the mean arterial pressure (MAP) was monitored continuously. The elimination half-life (t 1/2) of S15NALB from rat plasma was determined to be 4.1 min (t 1/2 α) and 9.4 min (t 1/2 β). S15NALB (125 nmol) produced long-lasting decreases in MAP (by 49% for 18 min). Thirty minutes after intravenous (i.v.) injection of S15NALB (125 nmol), repeated i.v. injection of l-cysteine or d-cysteine (10 μmol each) produced repeatedly potent (by 44–55%) but short-lasting (about 4 min) MAP falls. Intravenously administered GS15NO and S15NC (each 500 nmol) could not be isolated from rat blood. 15N-Labelled nitrite and nitrate were identified as the major metabolites of all investigated S-nitrosothiols in rat plasma. The results of this study suggest that in the rat S15NALB is a potent S-transnitrosylating agent and that the blood pressure-lowering effect of S15NALB and other S-nitrosothiols are mediated largely by l-cysteine via S-transnitrosylation to form S15NC that subsequently releases 15NO. Our results also suggest that S-transnitrosylation of the single reduced cysteine moiety of albumin by endogenous GSNO or SNC in blood is possible but does not represent an effective mechanism to produce SNALB in vivo. This stable-isotope dilution GC–MS technique is suitable to perform in vivo studies on SNALB using physiologically and pharmacologically relevant doses.
Keywords: Half-life; Nitric oxide; S-Nitrosothiols; S-Transnitrosylation; Affinity chromatography; Animal studies;

Quantitative determination of the 1,3-butadiene urinary metabolite 1,2-dihydroxybutyl mercapturic acid by high-performance liquid chromatography/tandem mass spectrometry using polynomial calibration curves by Mariella Carrieri; Giovanni Battista Bartolucci; Monica Livieri; Enrico Paci; Daniela Pigini; Renata Sisto; Federica Corsetti; Giovanna Tranfo (1388-1393).
1,3-Butadiene is used in the production of synthetic rubber and is also a widespread environmental pollutant, produced by car exhaust, heating and cigarette smoke. According to IARC it is probably carcinogenic to humans. A method was developed and validated for the quantification in human urine of 1,2-dihydroxybutyl mercapturic acid, a butadiene metabolite for which the American Conference of Governmental Hygienists suggests a biological exposure index of 2500 μg/L. Solid phase extraction was used for analyte extraction and HPLC–MS/MS for detection. The calibration range from 20 to 2500 μg/L required the use of polynomial calibration curves, and the performance of the analytical method was tested according to an international validation guideline. Accuracy was never less than 85%, precision always higher than 15% and the LOD 3.6 μg/L. The method was applied to 33 non-smokers, non-occupationally exposed to butadiene, and gave urinary concentrations between 16 and 599 μg/L.
Keywords: 1,3-Butadiene; DHBMA; Occupational exposure; Biological monitoring; HPLC–MS/MS; Method validation; Polynomial regression;

A method for the determination of prednisolone in human adipose tissue incubation medium has been developed, validated and used to support studies designed to measure the activity of 11β-hydroxysteroid dehydrogenase in human adipose tissue. After incubation, samples (80 μL) were extracted using Oasis HLB μElute SPE plates and the resulting extracts were analyzed using reversed-phase chromatography coupled to an Applied Biosystems Sciex PE API-4000 mass spectrometer with a TurboIonSpray® interface (400 °C). The method was validated over the calibration range of 0.5–100 ng/mL. Intraday precision and accuracy were 6.1% R.S.D. or less and within 6.3%, respectively. Interday precision and accuracy were 4.2% R.S.D. or less and within 3.6%, respectively. Extraction recovery of prednisolone was greater than 84% over the range of low to high quality control sample concentrations. The validated assay was used to support studies designed to estimate ex vivo 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme activity in human adipose tissue.
Keywords: Prednisolone; 11β-Hydroxysteroid dehydrogenase type 1; LC–MS/MS; μElute 96-well SPE; Human adipose tissue; Biomarker;

Epichlorohydrin (ECH) is an important industrial intermediate for the production of polymers and surface coatings. Animal experiments support the classification of ECH as a carcinogen, and a significant contribution to the cancer risk of ECH exposed humans has to be considered. Upon uptake, epichlorohydrin reacts with nucleophilic moieties of N- and S-containing macromolecules to form stable adducts, e.g. with haemoglobin. In this article, we describe a GC–tandem MS method for the quantitative analysis of the primary ECH adduct to the N-terminal amino acid of human haemoglobin, i.e. of N-(3-chloro-2-hydroxypropyl)valine (CHPV), using a globin labelled with d 5-ECH as the internal standard. Incubation of erythrocyte lysate from human blood with ECH or d 5-ECH yielded two reaction products, with CHPV being the major component. The GC–tandem MS method is based on a modified Edman degradation procedure with subsequent O-acetylation. The limits of detection and quantification of this method are 10 and 25 pmol/g globin, respectively. Intra- and inter-assay imprecision of the method was about 12 and 15%, respectively, and the mean recovery was 105 and 96% at the levels of 25 and 100 pmol of CHPV per g globin, respectively. The present study reports for the first time on the analysis of CHPV as a haemoglobin adduct of ECH using GC–tandem MS and a stable-isotope labelled internal standard. By this method we quantified haemoglobin adducts of ECH in the blood of subjects potentially exposed to ECH after a freight train accident. Our study points to CHPV in human haemoglobin as a possible biomarker for epichlorohydrin exposure.
Keywords: Epichlorohydrin; Biomonitoring; Deuterium-labelling; GC–tandem MS; Globin; Protein adducts;