Journal of Chromatography B (v.883-884, #C)
Editorial Board (i).
LC–MS/MS in clinical chemistry by Michael Vogeser; Christoph Seger (1-2).
10 years of MS instrumental developments – Impact on LC–MS/MS in clinical chemistry by Markus Himmelsbach (3-17).
The combination of liquid chromatography and mass spectrometry (LC–MS) is a powerful and indispensable analytical tool that is widely applied in many areas of chemistry, medicine, pharmaceutics and biochemistry. In this review recent MS instrumental developments are presented as part of a special issue covering various aspects of liquid chromatography tandem mass spectrometry (LC–MS/MS) in clinical chemistry. Improvements, new inventions as well as new combinations in ion source technology are described focusing on dual or multimode sources and atmospheric pressure photoionization (APPI). Increasing demands regarding sensitivity, accuracy, resolution and both quantitation and identification guarantee on-going improvements in mass analyzer technology. This paper discusses new hybrid MS instruments that can perform novel scan modes as well as high-resolution mass spectrometers (HRMS) that finally seem to be able to overcome, or at least significantly reduce, their weaknesses in quantitative applications. Ion mobility-mass spectrometry (IMMS) itself is not an invention of the last 10 years, but a lot of progress was made within the last decade that reveals the potential benefits of this combination. This is clearly reflected by the increased number of commercially available instruments and the various designs of IMMS are covered in detail in this review. Selected applications for all these instrumental developments are given focusing on the perspective of clinical chemistry.
Keywords: Liquid chromatography; Tandem mass spectrometry; High-resolution mass spectrometry; Ion mobility mass spectrometry; Atmospheric pressure photoionization; Quadrupole time-of-flight; Orbitrap; Review;
The role of liquid chromatography–tandem mass spectrometry in the clinical laboratory by Johannes M.W. van den Ouweland; Ido P. Kema (18-32).
Liquid chromatography coupled to mass spectrometry (LC–MS/MS) is increasingly used as a routine methodology in clinical laboratories for the analysis of low molecular weight molecules. The high specificity in combination with high sensitivity and multi-analyte potential makes it an attractive complementary method to traditional methodology used for routine applications. Its strength and weaknesses in this context will be discussed and examples of successful clinical applications will be given. For LC–MS/MS to truly fulfil its promise in clinical diagnosis, the prerequisite steps being sample pre-treatment, chromatographic separation and detection by selected reaction monitoring must become more integrated as they are in conventional clinical analysers. The availability of ready-to-use reagents kits, eliminating efforts needed for method development and extensive validation, are likely to contribute to a wider acceptance of LC–MS/MS in clinical laboratories. Growing applicability of LC–MS/MS in the clinical laboratory field is expected from quantitative protein analysis.
Keywords: Liquid chromatography; Mass spectrometry; Clinical chemistry;
Therapeutic drug monitoring and LC–MS/MS by Joanne E. Adaway; Brian G. Keevil (33-49).
LC–MS/MS is an increasingly important tool in therapeutic drug monitoring as it offers increased sensitivity and specificity compared to other methods, and may be the only viable method for quantifying drugs without natural chromophores or fluorophores. The choice of sample preparation method, column technology, internal standard and mass spectrometric conditions is important to ensure accurate drug measurement and to avoid interference from matrix effects and drug metabolites. LC–MS/MS is a more involved technique than automated immunoassays, but technological advances such as the development of pipetting robots and online solid phase extraction mean that LC–MS/MS is becoming an attractive and convenient method for therapeutic drug monitoring in clinical laboratories.
Keywords: Therapeutic drug monitoring; Liquid chromatography–tandem mass spectrometry;
The coming of age of liquid chromatography coupled to tandem mass spectrometry in the endocrinology laboratory by Valdemir Melechco Carvalho (50-58).
Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) has been rapidly incorporated in the routine of the endocrinology laboratory. Most endocrinologists are aware of the benefits afforded by this technique and tandem mass spectrometers are clearly no longer a mere research method but an important tool widely used for diagnosis. In the last 15 years, LC–MS/MS has replaced techniques such as immunoassay and HPLC for the analysis of hormones because it provides higher specificity and good sensitivity. Also, it permits simultaneous measurement of several analytes and sample preparation and acquisition are fast and simple. Although several strategies based on LC–MS/MS have been described in the last 15 years, there is still room for improvement. The impact of matrix effects and isobaric interferences have been addressed by only a few studies, and standardization with reference materials is available for a limited number of analytes. This review summarizes the application of LC–MS/MS in analyzing three classes of hormones: steroids, derivatives of the aromatic amino acids, and peptides and proteins. The benefits and current limitations of LC–MS/MS will be discussed for these hormone categories.
Keywords: Liquid chromatography–mass spectrometry; Tandem mass spectrometry; Endocrinology; Steroid hormones; Amino acid derived hormones; Peptide and protein hormones; Assays;
LC–MS/MS for protein and peptide quantification in clinical chemistry by Manfred Rauh (59-67).
The LC-triple quadrupole mass spectrometer (LC–MS/MS) is an increasingly common tool in the clinical laboratory. Established applications include routine assays for detecting inborn errors of metabolism, and for monitoring therapeutic drugs and steroids. Peptides and proteins in biological matrices have traditionally been quantified by immunological methods such as RIA or ELISA. These methods have the drawback of being insufficiently selective, often not allowing differentiation between the peptide and its derivatives or degradation fragments. The improved robustness and sensitivity of LC–MS-based techniques provide reliable alternatives for peptide quantification. Mass spectrometry does not require specific antibody reagents and is a powerful tool for the study of posttranslational modifications (PTM). In addition, several studies have demonstrated the utility of selected reaction monitoring (SRM) assays using stable-isotope-labelled (tryptic) peptides for quantifying proteins in human serum. Peptide-based MS/MS is a relatively new development in the measurement of clinically significant proteins, offering cost effectiveness, high throughput, multiplexed analysis and quantification, with the potential for combining the measurement of small molecules, peptides and proteins on a single technology platform. Quantitative analysis of proteins and peptides by LC–MS/MS is becoming a practical technique for clinical laboratories. To move from the laboratories of highly skilled analysts to routine clinical diagnostic laboratories requires that a number of technical hurdles be overcome in regard to sensitivity, imprecision, accuracy and the sample handling necessary for clinical use.
Keywords: LC–MS/MS; SRM; MRM; Protein; Peptide;
LC–MS-based metabolomics in the clinical laboratory by Susen Becker; Linda Kortz; Christin Helmschrodt; Joachim Thiery; Uta Ceglarek (68-75).
The analysis of metabolites in human body fluids remains a challenge because of their chemical diversity and dynamic concentration range. Liquid chromatography (LC) in combination with tandem mass spectrometry (MS/MS) offers a robust, reliable, and economical methodology for quantitative single metabolite analysis and profiling of complete metabolite classes of a biological specimen over a broad dynamic concentration range. The application of LC–MS/MS based metabolomic approaches in clinical applications aims at both, the improvement of diagnostic sensitivity and specificity by profiling a metabolite class instead of a single metabolite analysis, and the identification of new disease specific biomarkers. In the present paper we discuss recent advances in method development for LC–MS/MS analysis of lipids, carbohydrates, amino acids and biogenic amines, vitamins and organic acids with focus on human body fluids. In this context an overview on recent LC–MS/MS based metabolome studies for cancer, diabetes and coronary heart disease is presented.
Keywords: Clinical application; Metabolomics; LC–MS/MS; Tandem mass spectrometry;
Chromatographic methods for the determination of therapeutic oligonucleotides by A. Cary McGinnis; Buyun Chen; Michael G. Bartlett (76-94).
Both DNA and RNA are being explored for their therapeutic potential against a wide range of diseases. As these new drugs emerge, new demands arise for the analysis and quantitation of these biomolecules. Pharmacokinetic and pharmacodynamic analysis requirements for drug approval place enormous challenges on the methods for analyzing these therapeutics. This review will focus on bioanalytical methods for DNA antisense and aptamers as well as small-interfering RNA (siRNA) therapeutics. Chromatography methods employing ultraviolet (UV), fluorescence and mass spectrometric (MS) detection along with matrix-assisted laser desorption/ionization (MALDI) will be covered. Sample preparation from biological matrices will be reviewed as well as metabolite analysis and identification. All of these techniques are important contributions toward oligonucleotide therapeutic development. They will also be important in microRNA (miRNA) biomarker discovery and RNomics in general, as more non-coding RNAs are inevitably discovered.
Keywords: Oligonucleotides; Ion exchange; Ion pair; LC–MS; DNA; RNA;
Successful and cost-efficient replacement of immunoassays by tandem mass spectrometry for the quantification of immunosuppressants in the clinical laboratory by Pierre-Olivier Hétu; Robert Robitaille; Bernard Vinet (95-101).
The purpose of this paper is to describe the implantation of mass spectrometry in replacement of immunoassays for the measurement of immunosuppressant drugs in the clinical setting, from scientific and financial perspectives. A straightforward, rapid, and economical method was developed for the simultaneous quantification of tacrolimus, sirolimus, and cyclosporine. Following a simple protein precipitation step, supernatants are injected on a small C18 guard cartridge and gradient elution of the immunosuppressants is performed in a total chromatographic run time of 2.25 min. Sodium adducts of the compounds and internal standards are quantified by electrospray tandem mass spectrometry. The method shows inter-assay impression of less than 10–15% for all compounds with good extraction efficiency (89–104%) and minimal matrix effects, except for sirolimus where ion suppression is more pronounced. The method correlates well with chemiluminescent microparticle immunoassays (on the Abbott Architect analyzer), although the immunoassay results are significantly higher than those obtained by HPLC–MS/MS. The transition from immunoassays to mass spectrometry was well received by the laboratory staff, and significant reductions in reagent costs have been realized (>$250,000 CAD per year). With these savings, the purchase and installation of two complete HPLC–MS/MS systems was completely financed in less than three years.
Keywords: Tacrolimus; Sirolimus; Cyclosporine A; Mass spectrometry;
Simultaneous measurement of cyclosporin A and tacrolimus from dried blood spots by ultra high performance liquid chromatography tandem mass spectrometry by Edward Hinchliffe; Joanne E. Adaway; Brian G. Keevil (102-107).
Cyclosporin A (CsA) and tacrolimus are immunosuppressant drugs principally used in solid organ transplant recipients. Therapeutic drug monitoring (TDM) of both drugs is essential to avoid toxicity related to overdosage, and transplant rejection from underdosage. This necessitates frequent hospital visits to phlebotomy services. Capillary blood sampling onto dried blood spots (DBS) provides numerous advantages to venous whole blood sampling, including the ability for patients to send DBS to the laboratory by post, significantly reducing the number of unnecessary hospital visits. We have developed a novel, simple and rapid method for the extraction and simultaneous UPLC–MS/MS measurement of both CsA and tacrolimus from DBS. The extraction method involved a simple 30 min hot solvent extraction with ultrasonication. Extract (10 μL) was injected onto a Waters Acquity UPLC column filter unit security frit, coupled to a Waters Acquity BEH C18 UPLC column, with methanolic mobile phase gradient elution. Eluant was connected to a Waters Quattro Premier XE tandem mass spectrometer operating in ES+ mode. We detected multiple reaction monitoring (MRM) transitions of m/z 1220 > 1203 and 1231.9 > 1215.1 for CsA and d12 CsA respectively which co-eluted at 1.30 min, and 821.6 > 768.5 and 809.6 > 756.5 for tacrolimus and ascomycin respectively which co-eluted at 1.17 min. Ion suppression was negligible. Mean recovery was 95.5% for CsA and 92.8% for tacrolimus. Limit of detection and limit of quantitation were both 8.5 μg/L for CsA, and 0.5 and 2.3 μg/L respectively for tacrolimus. The assay was linear up to 1500 μg/L for CsA (r 2 = 0.9999), and up to 50 μg/L for tacrolimus (r 2 = 0.9994). Mean intra assay imprecision, inter assay imprecision and bias were all <10% for both CsA and tacrolimus. DBS were stable for at least 14 days at room temperature. Comparison of the DBS UPLC–MS/MS method and the routine venous whole blood LC–MS/MS assay demonstrated good agreement between the two methods for both drugs. We have developed a simple and robust method for the extraction and simultaneous measurement of CsA and tacrolimus from DBS. The method will allow TDM of transplant recipients to proceed at home using capillary blood sampling.
Keywords: Cyclosporin A; Tacrolimus; Therapeutic drug monitoring; Dried blood spots; Liquid chromatography tandem mass spectrometry;
Therapeutic drug monitoring of tacrolimus by liquid chromatography–tandem mass spectrometry: Is it truly a routine test? by Paul J. Taylor; Michael E. Franklin; Chun-Hui Tai; Peter I. Pillans (108-112).
Therapeutic drug monitoring of tacrolimus by high-performance liquid chromatography–tandem mass spectrometry has become standard practice. We report on the long-term (4.5 years) use of one such method. Whole blood samples (25 μL) were treated with zinc sulphate (100 μL) and acetonitrile containing ascomycin (internal standard, 250 μL). A high-performance liquid chromatography–tandem mass spectrometer operating in positive ion mode with an electrospray interface was used. Chromatography was performed on a TDM C18 cartridge column (10 mm × 2.1 mm, 10 μm, Waters) using a switch gradient. A total of 4029 batches were analyzed for tacrolimus; this comprised of 81950 analyses of which 61027 were patient samples. Calibration curves (1.0–50 μg/L) were run on 1765 occasions (mean r 2 = 0.999; range r 2 = 0.988–0.999). Inter-batch accuracy and imprecision of the method (2.5, 12.5 and 30.0 μg/L), when in routine use, was 97.6–98.5% and <8.0%, respectively (n = 4031). Evaluation of the method against other methods in an external quality control scheme revealed good agreement by linear regression analysis (y = 0.924x + 0.196, r 2 = 0.985). The percentage difference between our results and that of all methods revealed a mean bias of −6.3% and a range of −33.3% to 11.1%. During the evaluation period, four batch failures occurred (0.1% failure rate) and greater than 1000 samples per analytical column was achieved. In conclusion, the described method is ideally suited as a routine test for tacrolimus in the clinical setting.
Keywords: Tacrolimus; Therapeutic drug monitoring; HPLC; Tandem mass spectrometry;
A high-throughput U-HPLC–MS/MS assay for the quantification of mycophenolic acid and its major metabolites mycophenolic acid glucuronide and mycophenolic acid acyl-glucuronide in human plasma and urine by Jacek Klepacki; Jelena Klawitter; Jamie Bendrick-Peart; Bjorn Schniedewind; Svenja Heischmann; Touraj Shokati; Uwe Christians; Jost Klawitter (113-119).
Mycophenolic acid (MPA) is used as an immunosuppressant after organ transplantation and for the treatment of immune diseases. There is increasing evidence that therapeutic drug monitoring and plasma concentration-guided dose adjustments are beneficial for patients to maintain immunosuppressive efficacy and to avoid toxicity. The major MPA metabolite that can be found in high concentrations in plasma is MPA glucuronide (MPAG). A metabolite usually present at lower concentrations, MPA acyl-glucuronide (AcMPAG), has been implicated in some of the adverse effects of MPA. We developed and validated an automated high-throughput ultra-high performance chromatography–tandem mass spectrometry (U-HPLC–MS/MS) assay using liquid-handling robotic extraction for the quantification of MPA, MPAG, and AcMPAG in human EDTA plasma and urine. The ranges of reliable response were 0.097 (lower limit of quantitation) to 200 μg/mL for MPA and MPAG and 0.156–10 μg/mL for AcMPAG in human urine and plasma. The inter-day accuracies were 94.3–104.4%, 93.8–105.0% and 94.4–104.7% for MPA, MPAG and AcMPAG, respectively. Inter-day precisions were 0.7–7.8%, 0.9–6.9% and 1.6–8.6% for MPA, MPAG and AcMPAG. No matrix interferences, ion suppression/enhancement and carry-over were detected. The total assay run time was 2.3 min. The assay met all predefined acceptance criteria and the quantification of MPA was successfully cross-validated with an LC–MS/MS assay routinely used for clinical therapeutic drug monitoring. The assay has proven to be robust and reliable during the measurement of samples from several pharmacokinetics trials.
Keywords: Mycophenolic acid; Mycophenolic acid glucuronide; Mycophenolic acid acyl-glucuronide; U-HPLC–MS/MS; Immunosuppressant;
Development and optimization of simplified LC–MS/MS quantification of 25-hydroxyvitamin D using protein precipitation combined with on-line solid phase extraction (SPE) by Denis Thibeault; Nicolas Caron; Rose Djiana; Richard Kremer; David Blank (120-127).
25-Hydroxyvitamin D, the most useful marker of the vitamin D status of an individual, has seen an exponential growth of its routine measurement in recent years. Several methods are currently offered but the most specific is LC–MS/MS. However, the routine use of this technique in the clinical laboratory makes it essential to improve key steps of this method for high throughput delivery. Importantly, the preanalytical steps of this assay and the efficacy of the separation system need to be optimized prior to MS detection. In this report we replaced the standard and time consuming liquid–liquid extraction method of vitamin D metabolites with hexane (LLE) combined with centrifugation (LLE/centrifugation) by a simpler protein precipitation with extraction (PPE) in acetonitrile combined with a fast separation process using a 96-well plate filtration system (PPE/filtration). This rapid extraction was then followed by an on-line solid phase extraction (SPE) using a selective chromatographic separation. We also optimized the operational and consumable costs, by using an inexpensive guard column as a trapping column to significantly enhance the lifespan of the analytical column two to three times as compared to conventional chromatography. The LC–MS/MS technique permits the measurement of both 25-hydroxyvitamin D2 (25-OH D2) and the 25-hydroxyvitamin D3 (25-OH D3) metabolites in electrospray ionization (ESI) mode. The chromatographic system consisted of a 2.1 mm × 50 mm C18 3.5 μM column with a 2.1 mm × 20 mm C18 3.5 μM guard column connected with two 6 ports switching valves. Quantifications were done using the isotopic dilution technique with hexadeutered 25-OH D3 and 25-OH D2.The ion suppression problem with phospholipids was also evaluated and optimized to minimize this effect through the chromatography process and the on-line SPE trapping. Calibration curves were prepared by diluting a commercial high calibrator Chromsystems (München, Germany) with either pure triple stripped blank serum or diluted in 6% phosphate buffer saline at pH 7.2. Linearity was tested up to 160 nmol/L for 25-OH D3 and 75 nmol/L for 25-OH D2. Low limit of quantification (LLOQ) were established at 3 nmol/L for 25-OH D2 and 4 nmol/L for 25-OH D3. Inter-assay and intra-assay precision (CV%) was determined using 3 levels of commercial controls (Utak, CA, USA) for 25-OH D2 and 25-OH D3. Results obtained for intra-assay and inter-assay precision (CV%) were 1.1–3.4% and 5–8.9% respectively for the PPE/centrifugation technique and 2.0–3.1% and 4.6–6.6% for the PPE/filtration technique. Accuracy was estimated with the same commercial controls: % bias was −11.2 to 4.9% with PPE/centrifugation and −3.2 to 6.1% with PPE/filtration. 25-OH D2 and 25-OH D3 concentrations in human serum with LLE were compared to the new extraction methods using either PPE/centrifugation or PPE/filtration. Correlations comparing the two methods revealed a slope approximately 1.0 ± 0.3 with R ≥ 0.98 with a bias < 1 nmol/L. In summary, the new LC–MS/MS method described in this report using an on-line SPE technique with a simple off-line pre-treatment is faster, cost-effective, more reliable and more robust than current and widely used LLE/centrifugation methods coupled with LC–MS/MS.
Keywords: 25-Hydroxyvitamin D; LC–MS/MS; SPE on-line; Method comparison;
Quantification of the Fabry marker lysoGb3 in human plasma by tandem mass spectrometry by Ralf Krüger; Andreas Tholey; Thomas Jakoby; Rita Vogelsberger; René Mönnikes; Heidi Rossmann; Michael Beck; Karl J. Lackner (128-135).
Morbus Fabry is a hereditary metabolic disorder with low prevalence and late clinical manifestation. A defect in the α-galactosidase gene leads to lysosomal accumulation of the glycolipid globotriaosylceramide (Gb3). Gb3 may be used for monitoring of enzyme replacement therapy (ERT), but diagnostic sensitivity is limited. Recently, globotriaosylsphingosine (lysoGb3) was introduced as a promising new marker with significantly better sensitivity . For Fabry diagnosis, clinical studies and possible therapy monitoring, we established a fast and reliable LC–MS/MS assay for quantification of lysoGb3 in human plasma. Protein precipitation and glycolipid extraction from EDTA plasma was performed using acetone/methanol. Samples were analyzed by UPLC–MS/MS in MRM mode. In contrast to HPLC with fluorescence detection, the LC–MS/MS method requires no derivatization, less sample preparation and less instrument analysis time (<3 min). As internal standard (ISTD), a glycine derivative of lysoGb3 was synthesized, and the product was purified by HPLC. ISTD properties such as polarity (affecting extraction and elution), ionization and fragmentation pathway were almost identical compared to the analyte. The new LC–MS/MS assay for the Fabry marker lysoGb3 shows good performance and allowed for better discrimination between Fabry patients and controls than Gb3.
Keywords: Morbus Fabry; Plasma; LysoGb3; Globotriaosylsphingosine; UPLC–MS/MS; Internal standard;
Flow injection tandem mass spectrometric measurement of ceramides of multiple chain lengths in biological samples by Jie Chen; Srinivas B. Narayan; Aimee L. Edinger; Michael J. Bennett (136-140).
A method is presented for the measurement of ceramide species in biological fluids using flow injection tandem mass spectrometry. Ceramides are important signaling compounds in a number of cell:cell interactions including apoptosis and neurodegeneration. Because of the large number of potential fatty acid constituent moieties on ceramide molecules, a method which accurately distinguishes different chain-length species was required. The present method does not require HPLC separation and is designed to be applicable to high throughput analysis required for clinical studies. We provide a reference range for all measurable ceramide species in normal human plasma and an example of the utility of the assay in providing biomarkers in an in vitro apoptotic cell death study using murine hematopoietic cells treated with daunorubicin.
Keywords: Ceramides; Electrospray tandem spectrometry; Cell death studies; Biomarkers;
Quantification of bioactive sphingo- and glycerophospholipid species by electrospray ionization tandem mass spectrometry in blood by Gerhard Liebisch; Max Scherer (141-146).
Bioactive glycerophospho- and sphingolipids species are involved in the regulation of numerous biological processes and implicated in the pathophysiology of various diseases. Here we review electrospray ionization tandem mass spectrometric (ESI-MS/MS) methods for the analysis of these bioactive lipid species in blood including lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), bis(monoacylglycero)phosphate (BMP), ceramide (Cer), sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC). Beside direct tandem mass spectrometric and liquid chromatography coupled approaches, we present an overview of concentrations of these bioactive lipids in plasma. The analytical strategies are discussed together with aspects of sample preparation, quantification and sample stability.
Keywords: Liquid chromatography tandem mass spectrometry (LC–MS/MS); Lysophospholipids; Lipidomics;
Exploratory study of proteins in urine of patients with histoplasma antigenuria by Mark M. Kushnir; David K. Crockett; Joann L. Cloud; Edward R. Ashwood; Alan L. Rockwood (147-154).
Disseminated histoplasmosis is an invasive fungal infection that can be fatal in patients with weak immune system. The goal of our exploratory study was to evaluate differences in urinary protein profiles among samples of healthy individuals, patients with proteinuria (PRU), and histoplasma antigenuria (HIS), and to identify physiological pathways associated with the excreted proteins. Urine samples were depleted of abundant proteins, deglycosylated, digested with trypsin, fractionated and analyzed by nano-LC-QTOF. The total number of human proteins identified in the samples was 117, of which 20 and 23 were unique to the samples from patients with PRU and HIS, respectively. Pathway analysis of proteins identified in samples of PRU and HIS patients suggested increased levels of proteins associated with acute response signaling, coagulation system, prothrombin activation, glucocorticoid regulation and the lipid antigen presentation signaling pathway networks. The obtained data provide information on protein expression associated with HIS, and suggest that further more rigorous studies aimed at the identification of proteins associated with proteinuria of different causes are feasible.
Keywords: Histoplasmosis; Mass spectrometry; Proteins; Proteinuria; Pathway analysis;
Reference intervals for orotic acid in urine, plasma and dried blood spot using hydrophilic interaction liquid chromatography–tandem mass spectrometry by Oceania D’Apolito; Daniela Garofalo; Giancarlo la Marca; Antonio Dello Russo; Gaetano Corso (155-160).
Orotic acid (OA), a marker of hereditary orotic aciduria, is usually used for the differential diagnosis of some hyperammonemic inherited defects of urea cycle and of basic amino acid transporters. This study was aimed to establish age related reference intervals of OA in urine, and for the first time in plasma, and dried blood spot (DBS) from 229 apparently healthy subjects aged from three days to 40 years. The quantification of OA was performed by a previously implemented method, using a stable isotope dilution with 1,3-[15N2]-orotic acid and hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS). The method has proved to be sensitive and accurate for a quantitative analysis of OA also in DBS and plasma. According to previous studies, urinary OA levels (mmol/mol of creatinine) decrease significantly with age. The upper limits (as 99th %ile) were of 3.44 and 1.30 in groups aged from three days to 1 year (group 1) and from 1 year to 12 years (group 2), respectively; in teenagers (from 13 to 19 years; group 3) and adults (from 20 to 40 years; group 4) urinary levels became more stable and the upper limits were of 0.64 and 1.21, respectively. Furthermore, OA levels in DBS (μM) also resulted significantly higher in subjects of group 1 (upper limit of 0.89) than in subjects of groups 2, 3 and 4 (upper limits of 0.24, 0.21, and 0.29, respectively). OA levels in plasma (μM) were significantly lower in subjects of group 3 (upper limit of 0.30) than in subjects of groups 1, 2, and 4 (upper limits of 0.59, 0.48, and 0.77, respectively). This method was also employed for OA quantification in plasma and DBS of 17 newborns affected by urea cycle defects, resulting sensitive and specific enough to screen these disorders.
Keywords: Reference intervals; Orotic acid; Dried blood spot; Tandem mass spectrometry;
Simultaneous UPLC–MS/MS quantification of the endocannabinoids 2-arachidonoyl glycerol (2AG), 1-arachidonoyl glycerol (1AG), and anandamide in human plasma: Minimization of matrix-effects, 2AG/1AG isomerization and degradation by toluene solvent extraction by Alexander A. Zoerner; Sandor Batkai; Maria-Theresia Suchy; Frank-Mathias Gutzki; Stefan Engeli; Jens Jordan; Dimitrios Tsikas (161-171).
Analysis of the endocannabinoid (EC) system's key molecules 2-arachidonoyl glycerol (2AG) and arachidonoyl ethanolamide (anandamide, AEA) is challenging due to several peculiarities. 2AG isomerizes spontaneously to its biologically inactive analogue 1-arachidonoyl glycerol (1AG) by acyl migration and it is only chromatographically distinguishable from 1AG. Matrix-effects caused primarily by co-extracted phospholipids may further compromise analysis. In addition, 2AG and 1AG are unstable under certain conditions like solvent evaporation or reconstitution of dried extracts. We examined effects of different organic solvents and their mixtures, such as toluene, ethyl acetate, and chloroform-methanol, on 2AG/1AG isomerisation, 2AG/1AG stability, and matrix-effects in the UPLC–MS/MS analysis of 2AG and AEA in human plasma. Toluene prevented, both, 2AG isomerisation to 1AG and degradation of 2AG/1AG during evaporation. Toluene extracts contain only 2% of matrix-effect-causing plasma phospholipids compared to extracts from the traditionally used solvent mixture chloroform–methanol. Toluene and all other tested organic solvents provide comparable 2AG and AEA extraction yields (60–80%). Based on these favourable toluene properties, we developed and validated a UPLC–MS/MS method with positive electrospray ionization (ESI+) that allows for simultaneous accurate and precise measurement of 2AG and AEA in human plasma. The UPLC–MS/MS method was cross-validated with a previously described fully-validated GC–MS/MS method for AEA in human plasma. A close correlation (r 2 = 0.821) was observed between the results obtained from UPLC–MS/MS (y) and GC–MS/MS (x) methods (y = 0.01 + 0.85x). The UPLC–MS/MS method is suitable for routine measurement of 2AG and AEA in human plasma samples (1 mL) in clinical settings as shown by quality control plasma samples processed over a period of 100 days. The UPLC–MS/MS method was further extended to human urine. In urine, AEA was not detectable and 2AG was detected in only 3 out of 19 samples from healthy subjects at 160, 180 and 212 pM corresponding to 12.3, 14.5 and 9.9 pmol/mmol creatinine, respectively.
Keywords: AEA; 2AG; Clinical studies; Endocannabinoids; Isomerisation; Matrix-effects; Plasma; Quantification; Tandem mass spectrometry;
Protein characterization by LC–MS/MS may be required for the DNA identification of a fusion hemoglobin: The example of Hb P-Nilotic by Isabelle Zanella-Cleon; Frédéric Delolme; Philippe Lacan; Caroline Garcia; Isabelle Vinatier; Alain Francina; Philippe Joly (172-176).
DNA analysis is currently the easiest way to identify a hemoglobin variant in most cases. Nevertheless, in case of complex gene rearrangements, mass spectrometry studies may be required to orientate the DNA diagnosis. The present report shows the use of mass spectrometry techniques prior to DNA analysis for the identification of the rare P-Nilotic fusion hemoglobin. Complete protein analysis is performed by liquid chromatography–tandem mass spectrometry on the abnormal globin chain isolated by reversed-phase liquid chromatography.
Keywords: Hemoglobin variant; Fusion hemoglobin; Anti-Lepore; LC–MS/MS;
Determination of benzodiazepines in ante-mortem and post-mortem whole blood by solid-supported liquid–liquid extraction and UPLC–MS/MS by E.N. Sauve; M. Langødegård; D. Ekeberg; Å.M.L. Øiestad (177-188).
A solid-supported liquid–liquid extraction ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the determination of benzodiazepines commonly found in Norway, for use in cases with suspected driving impairment and autopsy cases by analysis of human whole blood samples. The following compounds were included: alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, lorazepam, midazolam, nitrazepam, nordiazepam (metabolite of diazepam), oxazepam and phenazepam. Aliquots of 500 μL whole blood were added 500 μL of borate buffer pH 11 and extracted by solid-supported liquid–liquid extraction on ChemElut® columns using three times 2.5 mL of methyl tert-butyl ether. Deuterated analogues were used as internal standards (IS) for all analytes, except for midazolam, phenazepam and bromazepam which had no commercially available deuterated analogues at the time the method was developed, and therefore used diazepam-d5, flunitrazepam-d7 and nitrazepam-d5, respectively. The analytes were separated using UPLC with a 2.1 × 100 mm BEH C18-column, 1.7 μm particle size, and quantified by MS/MS using multiple reaction monitoring (MRM) in positive mode. Two transitions were used for the analytes and one transition for the IS. The run time of the method was 8 min including equilibration time. The concentrations of the benzodiazepines in the method span a broad range varying from the lowest concentration of 0.005 μM for flunitrazepam to the highest of 20 μM for oxazepam. The calibration curves of extracted whole blood standards were fitted by second-order calibration curves weighted 1/x, with R 2 values ranging from 0.9981 to 0.9998. The intermediate precision had a CV (%) ranging between 2 and 19%. Recoveries of the analytes were from 71 to 96%. The LLOQs for the analytes varied from 0.0006 to 0.075 μM and the LODs from 0.005 to 3.0 nM. Matrix effects were studied by post extraction addition and found to be between 95 and 104% when calculated against an internal standard. A comparison with two other LC–MS methods was performed during method validation. Good correlation was seen for all analytes. The method has been running on a routine basis for several years, and has proven to be very robust and reliable with good results for external quality samples. The method also meets the requirements of the legislative limits for driving under the influence of non-alcohol drugs to be introduced in the Norwegian legislative system from 2012.
Keywords: Benzodiazepines; Whole blood; Quantitative analysis; Solid-supported liquid–liquid extraction; Ultra performance liquid chromatography tandem mass spectrometry;
Online extraction toxicological MS(n) screening system for serum and heparinized plasma and comparison of screening results between plasma and urine in the context of clinical data by Daniel M. Mueller; Katharina M. Rentsch (189-197).
The two main matrices for screening are urine or serum and heparinized plasma. Whereas urine has the advantage of usually higher concentrations and longer detection windows, serum or heparinized plasma represent the current systemic drug exposure of a patient.An online extraction LC–MS n method using a MS2 and MS3 spectral library for the identification of substances has been developed and validated to screen serum and heparinized plasma. Extraction was performed by online turbulent flow chromatography under alkaline conditions. Chromatographic separation was achieved using a phenyl/hexyl column with acidic eluents. For detection, a linear ion trap, equipped with an APCI interface, was used and the different compounds were identified using a MS2 and MS3 spectral library containing 453 compounds.From 47 patients, urine and heparinized plasma samples were analyzed and the results compared.The validation of the method gave satisfactory results. Only 3% of the compounds showed a matrix effect > 10% in serum. For all other substances and heparinized plasma, the quantitative matrix effect was <10%. 78% of the compounds where a therapeutic range was described in the literature had a limit of identification below the therapeutic range in heparinized plasma and 77% in serum, respectively.In urine and heparinized plasma samples, a total of 168 substances (identified as 86 different compounds) could be identified. In 20 out of 47 cases (43%), the results were identical. On a substance level, the agreement between urine and heparinized plasma was in average 71% with a range of 0–100%.The presented method allows a fast identification of 453 substances in serum and heparinized plasma. If plasma or serum is used for toxicological screening, the current systemic exposure of a patient can be monitored.
Keywords: Toxicological screening; Clinical toxicology; Online extraction; Turbulent flow chromatography; LC–MS;
Formation and characterization of covalent guanosine adducts with electrochemistry—liquid chromatography–mass spectrometry by Sabine Plattner; Robert Erb; Florian Pitterl; Hendrik-Jan Brouwer; Herbert Oberacher (198-204).
Chemicals can interact with the genetic material giving rise to the formation of covalent adducts. These alterations can lead to adverse consequences, including cancer, reproductive impairment, development anomalies, or genetic diseases. In search for an assay allowing identification of hazardous compounds that might form covalent adducts with nucleic acids, electrochemistry (EC)/liquid chromatography (LC)/mass spectrometry (MS) is presented. EC/LC/MS is a purely instrumental approach. EC is used for oxidative activation, LC for the fractionation of the reaction mixture, and MS for the detection and characterization of the reaction products. To test the system capabilities, we investigated the formation of covalent adducts produced by guanosine and acetaminophen (APAP). Electrochemical activation of mixtures of guanosine and APAP gave rise to the formation of four isomers of (guanosine + APAP-2H). Mass voltammograms as well as dose–response-curves were used to obtain insights in the mechanism of adduct formation. These experiments revealed that a mechanism involving radical intermediates is favored. The initial step of adduct formation is the conversion of both APAP and guanosine into radicals via one-electron–one-proton reactions. Among different competing reaction pathways, the generated radical intermediates undergo intermolecular reactions to form covalent adducts between guanosine and APAP.
Keywords: Electrochemistry; Mass spectrometry; Nucleic acids; Liquid chromatography; DNA adduct; DNA damage;