Metabolites In Biological Fluids Research Articles

An improved mass spectrometry-based measurement of NO metabolites in biological fluids

Assessment of NO metabolism in vivo relies on the accurate measurement of its metabolites nitrite (NO2−), nitrate (NO3−), and nitrosothiols (RSNOs) in biological fluids. We report a sensitive method to simultaneously determine NO2− and NO3− in biological matrixes. Tetraoctylammonium was used to catalyze the complete conversion of NO2− and NO3− to stable pentafluorobenzyl (PFB) derivatives directly from aqueous acetone medium before gas chromatography and negative-ion chemical ionization mass spectrometry (GC/NICI/MS). This catalyst dramatically improved the yield of PFB derivatives for NO2− (4.5 times) and NO3− (55 times) compared to noncatalyzed derivatization methods. Analysis was performed using 15N-labeled internal standards by selected-ion monitoring at m/z 46 for fragment NO2− and m/z 47 for its isotope analogue, 15NO2−, and m/z 62 for NO3− and m/z 63 for 15NO3−. This method allowed specific detection of both PFB derivatives over a wide dynamic range with a limit of detection below 4.5 pg for NO2− and 2.5 pg for NO3−. After the specific conversion of RSNOs by HgCl2 to NO2−, this GC/NICI/MS analysis was used to measure RSNOs in plasma. A further comparison with the widely used tri-iodide chemiluminescence (I3−-CL) assay indicated that the GC/MS assay validated the lower physiological RSNO and nitrite levels reported using I3−-CL detection compared with values obtained using UV–photolysis methods. Plasma levels of RSNOs determined by GC/MS and I3−-CL were well correlated (r = 0.8). The improved GC/MS method was successfully used to determine the changes in plasma, urinary, and salivary NO2− and NO3− as well as plasma RSNOs in humans after either a low-NO3− or a high-NO3− meal.

A rapid and accurate UPLC/MS/MS method for the simultaneous determination of zolpidem and its main metabolites in biological fluids and its application in a forensic context

Zolpidem (ZPD) is an imidazopyridine derivative used as a new type of hypnotic and is commonly used in drug-facilitated crimes. A rapid, sensitive, and specific ultra-performance liquid chromatography–tandem mass spectrometry (UPLC/MS/MS) assay method for the simultaneous determination of zolpidem and its main metabolites zolpidem 6-carboxylic acid (ZCA) and zolpidem phenyl-4-carboxylic acid (ZPCA) in biological fluids was developed and validated. Aliquots of 0.1mL blood or urine specimens were used for the analysis, and zolpidem and its metabolites were extracted in a single step using acetonitrile (containing 0.1% formic acid) precipitation. The supernatant was then dried, and 100μL methanol was added. The separation was performed on an Acquity™ UPLC HSS T3 (100mm×2.1mm, 1.8μm) analytical column by API 4000Q ultra-performance liquid chromatography–tandem mass spectrometry. Positive ionisation tandem MS detection in the multiple reaction monitoring (MRM) mode was used. The mobile phases consisted of either acetonitrile or water containing 20mmol/L ammonium acetate and 0.1% formic acid, and the flow rate was 0.5mL/min. The chromatographic separation time was 4min, and calibration curves for human blood were generated over the range of 0.1–300ng/mL for ZPD, 0.1–500ng/mL for ZPCA and 0.1–200ng/mL for ZCA. For urine, the linear range was 0.1–600ng/mL for ZPD and ZPCA, and 0.1–300ng/mL for ZCA. The limit of detection was 0.05ng/mL and the limit of quantitation was 0.1ng/mL for ZPD, ZCA and ZPCA. The linear correlation coefficients were greater than 0.9995. Both the inter-day and intra-day precisions were less than 15%, the recoveries were in the range of 70.0–98.3%, the matrix effects were approximately 79.5–99.0%, and the process efficiency was between 60.7% and 94.4%. This method allowed for the determination of zolpidem and its metabolites in human blood and urine and may be applied to forensic toxicological analyses.

JS1-5 - Pharmaco-Metabolomics for Anticancer Drugs

ABSTRACT Metabolomics is a biochemical phenotype of cellular events after gene expression, transcription and translation, and recently it has been included as a new ‘omics’ science, as a new field toward the understanding of global system biology. Since there are numerous networks with thousands of metabolites in human cells, a key technology for metabolomics is the separation analysis of these abundant metabolites. The mass spectrometry (MS) plays a central role for identification and quantification of metabolites in biological fluids. LC-MS is useful for studying hydrophobic metabolites, while the capillary electrophoresis (CE)-MS is a powerful method for analyzing charged compounds such as weak anions and cations. Cancer metabolomics is an emerging field in cancer biology. Cancer-specific metabolic reprogramming such as the Warburg effect is considered to facilitate cell growth and division. Several signaling pathways implicated in cell proliferation also regulate metabolic pathways that incorporate nutrients into biomass, rather than ATP production. We have firstly applied the CE-TOFMS metabolomic analysis to the pharmacology. Pharmaco-metabolomics investigates the drug action on the global metabolomic profiling and pathways. A comprehensive metabolomic fingerprint of cancer cells is expected to differentiate response or non-response to the drug therapy. Our recent findings regarding two metabolic inhibitors (5-FU and gemcitabine) are presented as examples. The metabolomic analyses can create new findings on cancer biology, pharmacology as well as potential biomarkers for drug action in the post-genome era.

INTERCELLULAR AND INTRACELLULAR TRAFFICKING OF NAD+ AND NAD+ PRECURSORS, INTERMEDIATE METABOLITES AND SIGNAL METABOLITES

Although being currently illustrated in Biochemistry textbooks, NAD+ metabolism is still largely undefined in its features. Specifically, enzymes involved in NAD+ biosynthesis and some of the enzymes involved in its utilization localize to distinct subcellular compartments of the same cell and, functionally, also to different cell types of the same organism. These findings lead to revolutionize current ideas. For instance, 1) NAD+ biosynthesis from several precursors (e.g., Nicotinamide, Nicotinic Acid, Nicotinamide mononucleotide, Nicotinamide riboside, Tryptophan, collectively defined Vit. B3) is a systemic yet segmentary process, whose individual steps may occur in different cells/tissues/organs. These activate a crosstalk via the exchange of intermediate metabolites in biological fluids and the eventual NAD+ biosynthesis takes place in selected cells able to utilize it in diverse, fundamental processes. Therefore, NAD+ metabolism is an organ- ismal process encompassing local events. 2) Utilization of NAD+ for regulation of cell functions involves the trafficking, both subcellular (autocrine) and intercellular (paracrine), of signal-metabolites including NAD+ itself and NAD+-derived second messengers, e.g. Cyclic ADP-ribose and ADP-ribose. This hitherto unrecognized trafficking involves a complex in-terplay of ectoenzymes (e.g. CD38), plasmamembrane receptors and related signal transduction pathways, equilibrative and concentrative transporters, ion channels, whose outcome is the fine control of intracellular Ca2+ homeostasis and of Ca2+-dependent cell functions. Further elucidation of compartmentation of NAD+ and more extensive identification of its precursors/metabolites is expected to unveil at the mechanistic level a number of physiological and pathological processes, e.g. aging and age-related diseases.

INTERCELLULAR AND INTRACELLULAR TRAFFICKING OF NAD+ AND NAD+ PRECURSORS, INTERMEDIATE METABOLITES AND SIGNAL METABOLITES

Although being currently illustrated in Biochemistry textbooks, NAD+ metabolism is still largely undefined in its features. Specifically, enzymes involved in NAD+ biosynthesis and some of the enzymes involved in its utilization localize to distinct subcellular compartments of the same cell and, functionally, also to different cell types of the same organism. These findings lead to revolutionize current ideas. For instance, 1) NAD+ biosynthesis from several precursors (e.g., Nicotinamide, Nicotinic Acid, Nicotinamide mononucleotide, Nicotinamide riboside, Tryptophan, collectively defined Vit. B3) is a systemic yet segmentary process, whose individual steps may occur in different cells/tissues/organs. These activate a crosstalk via the exchange of intermediate metabolites in biological fluids and the eventual NAD+ biosynthesis takes place in selected cells able to utilize it in diverse, fundamental processes. Therefore, NAD+ metabolism is an organ- ismal process encompassing local events. 2) Utilization of NAD+ for regulation of cell functions involves the trafficking, both subcellular (autocrine) and intercellular (paracrine), of signal-metabolites including NAD+ itself and NAD+-derived second messengers, e.g. Cyclic ADP-ribose and ADP-ribose. This hitherto unrecognized trafficking involves a complex in-terplay of ectoenzymes (e.g. CD38), plasmamembrane receptors and related signal transduction pathways, equilibrative and concentrative transporters, ion channels, whose outcome is the fine control of intracellular Ca2+ homeostasis and of Ca2+-dependent cell functions. Further elucidation of compartmentation of NAD+ and more extensive identification of its precursors/metabolites is expected to unveil at the mechanistic level a number of physiological and pathological processes, e.g. aging and age-related diseases.

Development of electrospray ionization tandem mass spectrometry methods for the study of a high number of urine markers of inborn errors of metabolism

Rapid and specific screening methods to detect abnormal metabolites in biological fluids are important for the diagnosis of many Inborn Errors of Metabolism (IEM). In Galicia (N.W. Spain), where newborn screening (NBS) has long used both blood and urine dried samples, an expanded NBS by tandem mass spectrometry (MS/MS) begun in July 2000 analyzing amino acids and acylcarnitines in blood. The purpose of this study is the development of methods to widen and to complement the present NBS with the study of the selected metabolites in urine. We studied and optimized the fragmentation of a total of 96 marking compounds of IEM, as well as 34 isotopically labeled internal standards (IS). The isobaric interferences were resolved with the use of alternative fragmentation in 14 of the 28 groups found. The methods were validated for 68 compounds following the recommendations of the NCCLS. We have developed electrospray ionization (ESI)- MS/MS methods in positive and negative ionization modes to detect selected metabolites in urine. The study was performed by direct injection of amino acids and acylcarnitines in positive mode, and organic acids, acylglycines, purines and pyrimidines in negative mode. Run times were 2.5 and 2.6 min, respectively, allowing the daily analysis of a high number of samples. The validated methods were proved effective for the simultaneous study of a large number of metabolites which are commonly present in urine samples and are used for detecting IEM. The evaluation was done by searching diagnostic profiles with multiple markers to increase sensitivity and specificity (e.g., acylcarnitines plus amino acids) or with specific urine markers (cystine, homogentisic acid, sialic acid, N-acetylaspartic acid, etc.).

Exclusive recognition of sarcosine in water and urine by a cavitand-functionalized silicon surface

A supramolecular approach for the specific detection of sarcosine, recently linked to the occurrence of aggressive prostate cancer forms, has been developed. A hybrid active surface was prepared by the covalent anchoring on Si substrates of a tetraphosphonate cavitand as supramolecular receptor and it was proven able to recognize sarcosine from its nonmethylated precursor, glycine, in water and urine. The entire complexation process has been investigated in the solid state, in solution, and at the solid-liquid interface to determine and weight all the factors responsible of the observed specificity. The final outcome is a Si-based active surface capable of binding exclusively sarcosine. The complete selectivity of the cavitand-decorated surface under these stringent conditions represents a critical step forward in the use of these materials for the specific detection of sarcosine and related metabolites in biological fluids.

Metabolomics, Metabonomics and Functional Nutrition: The Next Step in Nutritional Metabolism and Biotherapeutics

Metabonomics is the newest member of the convergent "-omics" family. This multi-parametric technique is used for studying metabolites in biological fluids for the purpose of metabolic profiling for biomarker and diagnostic applications, and to study the effects of environmental stimuli (diet, weight loss, exercise) and other therapeutic applications (lifestyle modification and drug effects) on the metabolome. Metabonomics has the advantage for defining biological phenotypes, but has a close relationship to investigation of the proteome, driven by advances in NMR and MS. The technique has proved highly informative in applications that include toxicology, pharmacology, and the biomedical sciences. This review elaborates on the paradigm shift that is emerging from this discovery process.

Integrating “omics” Technologies to Conceptualize Dynamic Antimicrobial Peptide Responses

Rarely in human disease can a single host factor be identified as the primary causal factor of a disease, syndrome, or disorder. Rather, the clinical manifestations that culminate in a symptomatic state are usually triggered by a perturbation of multiple factors derived from several distinct, but integrated, domains of cellular regulation. Host immune responses, such as antimicrobial peptide (AMP) production and regulation, are the result of interdependent dynamic interactions between host cells, extracellular milieu, and the external environment. In order to truly elucidate disease pathogenesis, it is imperative to consider the entire system at play from a global perspective, which is the promise of “omics” technology. The term “omics” was originally coined as a discipline in molecular biology that examines global sets of biological molecules (Micheel et al., 2012), which consequently stimulated an “Omics Revolution” throughout the scientific community. Genomics emerged following the sequencing of the human genome in the 1990s and early 2000s. This subsequently led to proteomics, which includes the entire complement of proteins and their related structure, modifications, and function. Pauling et al. (1971) proposed that estimating the abundance of metabolites in biological fluids may indicate the functional status of a specific biological system. Consequently, metabolomics has recently surfaced as an approach to comprehensively identify and quantify low molecular weight exogenous and endogenous metabolites and compounds in a biological system using high-throughput methods. Using 16S rRNA gene sequencing tools originally developed in the field of environmental microbiology, we can now generate a site-specific microbiome profile for any given patient. The Human Microbiome Project and numerous other projects aim to discern relationships between human-associated microbes and health and disease (Zoetendal et al., 2006). Such research has generated massive and complex data sets, which requires further advancement and refinement of bioinformatics and biostatistical applications. Often, the data is used to produce computational models to distinguish specific characteristics of a given population. Due to the unavoidable complexities and required statistical sophistication, few medical fields have developed clinically useful applications for the resulting data. Thus, AMPs are an ideal set of biological molecules to assess, since there biological role spans numerous areas of clinical research. Due to their diverse microbicidal and immunomodulatory functions (Steinstraesser et al., 2011), AMPs comprise a major aspect of the host innate immune response (Nakatsuji and Gallo, 2012). Alterations in AMP production and/or localization are associated with several cutaneous dermatoses. Patients diagnosed with atopic dermatitis and psoriasis ultimately develop epidermal barrier defects and microbial susceptibility, or a hyperproliferative/inflammatory epidermis, respectively (Schauber and Gallo, 2008; Schroder, 2011). The contributions of AMPs to tissue integrity and epithelial defense was also established in the brain (Schluesener et al., 2012; Williams et al., 2012), urinary tract (Saemann et al., 2007; Zasloff, 2007; Ali et al., 2009), gastrointestinal tract (Lisitsyn et al., 2012), and certain malignancies (Kesting et al., 2012; Scola et al., 2012). However, altered AMP regulation does not occur exclusively, but instead embodies dynamic transformations that emerge as a disturbed system. The integration of several different “omics” techniques may provide a more complete understanding of the diverse factors contributing to a specific disease state, responses to pharmacologic agents, or novel alternatives to current treatment modalities.

Mass Spectrometry-based Quantitative Analysis and Biomarker Discovery

Mass spectrometry-based quantitative analysis and biomarker discovery using metabolomics approach represent one of the major platforms in clinical fields including for the prognosis or diagnosis, assessment of severity and response to therapy in a number of clinical disease states as well as therapeutic drug monitoring (TDM). This review first summarizes our mass spectrometry-based research strategy and some results on relationship between cysteinyl leukotriene (cysLT), thromboxane (TX), 12-hydroxyeicosatetraenoic acid (12-HETE) and other metabolites of arachidonic acid and diseases such as atopic dermatitis, rheumatoid arthritis and diabetes mellitus. For the purpose of evaluating the role of these metabolites of arachidonic acid in disease status, we have developed sensitive determination methods with simple solid-phase extraction and applied in clinical settings. In addition to these endogenous compounds, using mass spectrometry, we have developed actually applicable quantitative methods for TDM. Representative example was a method of TDM for sirolimus, one of the immunosuppressant agents for a recipient of organ transplant, which requires rigorous monitoring of blood level. As we recognized great potential in mass spectrometry during these researches, we have become interested in metabolomics as the non-targeted analysis of metabolites. Now, established strategy for the metabolomics investigation applies to samples from cells, animals and humans to separate groups based on altered patterns of metabolites in biological fluids and to identify metabolites as potential biomarkers discriminating groups. We would be honored if our research using mass spectrometry would contribute to provide useful information in the field of medical pharmacy.

Identification of novel circulating coffee metabolites in human plasma by liquid chromatography–mass spectrometry

This study reports a liquid chromatography–mass spectrometry method for the detection of polyphenol-derived metabolites in human plasma without enzymatic treatment after coffee consumption. Separation of available standards was achieved by reversed-phase ultra performance liquid chromatography and detection was performed by high resolution mass spectrometry in negative electrospray ionization mode. This analytical method was then applied for the identification and relative quantification of circulating coffee metabolites. A total of 34 coffee metabolites (mainly reduced, sulfated and methylated forms of caffeic acid, coumaric acid, caffeoylquinic acid and caffeoylquinic acid lactone) were identified based on mass accuracy (<4 ppm for most metabolites), specific fragmentation pattern and co-chromatography (when standard available). Among them, 19 circulating coffee metabolites were identified for the first time in human plasma such as feruloylquinic acid lactone, sulfated and glucuronidated forms of feruloylquinic acid lactone and sulfated forms of coumaric acid. Phenolic acid derivatives such as dihydroferulic acid, dihydroferulic acid 4′- O-sulfate, caffeic acid 3′- O-sulfate, dimethoxycinnamic acid, dihydrocaffeic acid and coumaric acid O-sulfate appeared to be the main metabolites circulating in human plasma after coffee consumption. The described method is a sensitive and reliable approach for the identification of coffee metabolites in biological fluids. In future, this analytical method will give more confidence in compound identification to provide a more comprehensive assessment of coffee polyphenol bioavailability studies in humans.

The MS-MS determination of the fragmentation pathways of some substituted 1,2,3-benzotriazin-4-ones with possible applications in metabolic studies.

1,2,3-Benzotriazin-4-one derivatives are extensively used as herbicides, insecticides and nematicides. Incorporation of these compounds into the human food chain is a cause for concern, since their toxicity to man is well documented. This work describes the development of a data-base of classical 1,2,3-benzotriazin-4-one fragmentation patterns and consequent sequential neutral mass losses which allows the prediction of the presence of a triazinone derived compound. Positive and negative ion tandem mass spectrometry techniques were employed since they allow rapid and sensitive structural characterization of drugs and their metabolites in biological fluids through the unambiguous monitoring of daughter ions from pre-selected primary ions.

Historical Perspective on The Development and Evolution of Bioanalytical Guidance and Technology

Bioanalytical methods employed for the quantitative determination of drugs and their metabolites in biological fluids provide essential regulatory data for bioavailability, bioequivalence, pharmacokinetic and toxicokinetic studies. The quality of these studies is directly related to the underlying bioanalytical data. Data generated by a typical bioanalytical laboratory is submitted to not only the local regulatory agency, but also to multiple regulatory agencies worldwide. Many pharmaceutical companies and CROs are now performing bioanalytical work for global submissions and the regulatory agencies are often reviewing the bioanalytical work performed in other countries. The bioanalytical workplace has become global and therefore needs universal rules for quality and compliance of bioanalysis. This paper provides a historical perspective and insight into the development and evolution of the regulatory guidance for bioanalytical method validation and analysis of samples.

A Novel High Throughput Assay For Measuring Bacterial Adhesion Activity to Epithelial Cells

Research studies show cranberry helps to prevent UTIs by inhibiting E. coli from adhering to the uroepithelium that line up the urinary tract. Currently, many cell based anti‐adhesion assays have been developed, however, they are time consuming and lacking reproducibility due to the inherent limitations of the assays.In this new assay, human uroepithelial cells T24 are plated in multi‐well plates, grown to confluence, rendered nonviable but adhered to plates by UVC treatment. Testing samples are added to the cells in TB media, followed by adding E. coli 1161. After co‐culturing, plates are washed, bacteria that remain to be adhered to the cells are lysed, and their ATP contents are read by a plate reader.The NIST SRM 3283 Cranberry Extract is a chosen reference standard, the anti‐adhesion index of a sample is obtained by fitting the sample's inhibition degree to the dose response curve of the NIST reference. The results are expressed as SRM 3283 equivalent per mg or per mL. Various phytochemicals from cranberry including their metabolites in biological fluids, plant extracts, and juices were tested by this new assay. Our results indicate that when compared to other fruits, cranberry demonstrated the highest potency of anti‐adhesion activity. The assay is reliable, high throughput, and can be utilized to test a wide of variety of samples.

Identification of Coffee Metabolites in Human Plasma by Liquid Chromatography‐Mass Spectrometry

Chlorogenic acids are bioactive compounds found in coffee. They are of great interest for their health promoting effects but their bioavailability in human is not fully understood. Hence, it is essential to have strong analytical tools to accurately identify these bioactive compounds. Chlorogenic acids can be cleaved prior to absorption, resulting in the appearance of phenolic acids such as caffeic acid derivatives. A liquid chromatography-mass spectrometry method was developed for the identification of circulating forms of polyphenol-derived metabolites in human plasma after coffee consumption. Separation of metabolites was achieved by reversed-phase chromatography and detection was performed by high resolution MS in negative electrospray ionization mode. A total of 29 coffee metabolites were successfully identified based on co-chromatography (when standard available), mass accuracy and specific fragmentation pattern. Among them, phenolic acid derivatives such as dimethoxycinnamic acid, caffeic acid 3′O-sulfate, dihydroferulic acid appeared to be the main metabolites in human plasma. Only traces of chlorogenic acid conjugates were detected. This method is reliable for the efficient identification of native coffee metabolites in biological fluids. In future, this approach will give more confidence in metabolite identification to better assess coffee polyphenols bioavailability in humans.

Analyses of benzodiazepines and their metabolites in various biological matrices by LC-MS(/MS)

Benzodiazepines are among the most frequently prescribed drugs due to their sedative, hypnotic, anxiolytic, muscle relaxant and antiepileptic properties. Because of the high consumption of benzodiazepines worldwide, this class of drugs and their metabolites are frequently present in both clinical and forensic cases. For these reasons, the analysis of benzodiazepines and their metabolites in biological fluids is of great interest to clinicians and forensic toxicologists. This paper reviews procedures for multi-analyte single-stage (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) using different mass analyzers for the screening, identification and/or quantification of drugs, poisons and/or their metabolites in blood, plasma, serum or urine published since 2001. Basic information about the biosamples assayed, work-up, LC column, mobile phase, ionization type, mass spectral detection mode, matrix effects and validation data for each procedure is summarized. The feasibility of using LC-MS(/MS) techniques to identify and quantify benzodiazepines and their metabolites is also discussed.

Bioanalytical LC–MS of Therapeutic Oligonucleotides

Therapeutic oligonucleotides (OGNTs) are important biopharmaceutical drugs for the future, due to their ability to selectively reduce or knockout the expression of target genes. For the development of OGNTs, reliable and relatively high-throughput bioanalytical methods are required to perform the quantitative bioanalysis of OGNTs and their metabolites in biological fluids (e.g., plasma, urine and tissue). Although immunoaffinity methods, especially ELISA, are currently widely applied for this purpose, the potential of LC-MS in OGNT analysis is under investigation. Owing to its inherent ability to monitor the individual target OGNTs as well as their metabolites, LC-MS is now evolving into the method-of-choice for the bioanalysis of OGNTs. In this paper, the state-of-the-art of bioanalytical LC-MS of OGNTs and their metabolites in biological fluids is critically reviewed and its advantages and limitations highlighted. Finally, the future perspective of bioanalytical LC-MS, that is, lower detection levels and potential generic LC-MS methodology, is discussed.

Dissolution Method for Milnacipran Hydrochloride Capsules: Development, Validation, and Study of Changes in Dissolution Rate after Storage

A dissolution test for milnacipran hydrochloride capsules was developed and validated according to international guidelines. After selection of the best conditions, the method was validated using USP Apparatus 1 (baskets), 50-rpm rotation speed, 900 mL of 0.01 N HCl, and test time of 60 min. The drug released was determined by both LC–UV (PDA) and UV–D methods. The kinetic parameters of drug release (mathematical models, t80%, and dissolution efficiency) were investigated, and the stability of the dosage form was evaluated by analyzing changes in the dissolution rate of milnacipran hydrochloride capsules during storage at 40 °C and 75% RH for different periods. INTRODUCTION Dissolution testing can provide information not only on the rate and extent of drug absorption in the body but also on the effects of drug biopharmaceutical properties and formulation principles on the release properties of a pharmaceutical product (1). Therefore, in vitro dissolution tests are usually used to assess the lot-to-lot quality of a drug product, guide development of new formulations, an d ensure continued product quality and performance after certain changes such as formulation, manufacturing process, site of manufacture, and the scale-up of the manufacturing process (2). The dissolution procedure requires an apparatus, a dissolution medium, and test conditions that provide a method that is discriminating yet sufficiently rugged and reproducible for day-to-day operation and capable of being transferred between laboratories. With regard to stability, the dissolution test should appropriately reflect relevant changes in the drug product caused by temperature, humidity, photosensitivity, and other stresses over time (3). Milnacipran hydrochloride (MNC), [101152-94-7], C15H22N2O⋅HCl, molecular weight 282.81 g/mol (Figure 1), is a racemic mixture with the chemical name (±)-[1R(S),2S(R)]-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropanecarboxamide hydrochloride, and its solubility in water is 19 mg/mL (4–6). MNC is a selective serotonin and norepinephrine reuptake inhibitor (SNRI) indicated as an antidepressant and for the management of fibromyalgia. It shows preferential blockade of norepinephrine reuptake over serotonin and minimal activity at other receptors or transporters (6–8). MNC is well absorbed after oral administration with maximum concentrations reached within 2–4 h, and its absorption is not affected by food. It presents an absolute bioavailability of approximately 85–90% (9). The solubility and absolute bioavailability data for this drug classify it as Class I (high solubility and high permeability) based on the Biopharmaceutical Classification System. This is the case where the drug is well absorbed, and for immediate-release dosage forms that dissolve very rapidly, the absorption rate is controlled by the gastric emptying rate, and some correlation with dissolution rate is expected only if the dissolution is slower than gastric emptying (10). Methods for quantitation of milnacipran in combination with other antidepressants and their metabolites in biological fluids have been proposed (11–17). However, there is no compendial method to assay milnacipran hydrochloride in pharmaceutical dosage forms. A stability-indicating liquid chromatographic method with UV detection (LC–UV) and a second-order derivative UV spectroscopic method (UV–D) for quality control of milnacipran in capsules were developed and validated by the authors (18) according to guidelines (19–21). Therefore, the purpose of this work was to develop and validate a dissolution test for MNC in capsules (50 mg) based on its physicochemical characteristics and apply the LC–UV and UV–D methods to quantify the drug released from the capsules during the dissolution procedure. The kinetic parameters of drug release were investigated, and the stability of the dosage form was evaluated by analyzing changes in the dissolution rate of MNC capsules over time and in various storage conditions. EXPERIMENTAL Chemicals Milnacipran hydrochloride was purchased from Synfine Research (Canada). The pharmaceutical dosage form *Corresponding author. diss-18-03-06.indd 47 8/31/2011 1:38:15 PM dx.doi.org/10.14227/DT180311P47

Current medical research with the application of coupled techniques with mass spectrometry

SummaryThe most effective methods of analysis of organic compounds in biological fluids are coupled chromatographic techniques. Capillary gas chromatography/mass spectrometry (GC-MS) allows the most efficient separation, identification and quantification of volatile metabolites in biological fluids. Liquid chromatography-mass spectrometry (LC-MS) is especially suitable for the analysis of non-volatile and/or thermally unstable compounds. A major drawback of liquid chromatography-mass spectrometry is that no standard spectral libraries such as NIST and Wiley for GC-MS are available to facilitate the identification of unknown compounds. Moreover, the identification of potential new compounds, especially new biomarkers in LC-MS, is much more challenging than in GC-MS. Capillary electrophoresis coupled with mass spectrometry (CE-MS) has been widely used to characterize metabolomes. Capillary electrophoresis is a powerful technique for the separation of charged metabolites, offering high analyte resolution. The advantages of CE-MS are applicability for hydrophilic metabolites, robust separation efficiency and short duration of analysis.This review provides an overview of current chromatographic methods – gas chromatography – mass spectrometry, liquid chromatography – mass spectrometry and capillary electrophoresis-mass spectrometry – and their applications in current medical research. The focus is on the description of metabonomics research, strategies for biomarkers identification, medical diagnoses of diseases and research of drugs.

Electrochemical biosensors in pharmaceutical analysis

Given the increasing demand for practical and low-cost analytical techniques, biosensors have attracted attention for use in the quality analysis of drugs, medicines, and other analytes of interest in the pharmaceutical area. Biosensors allow quantification not only of the active component in pharmaceutical formulations, but also the analysis of degradation products and metabolites in biological fluids. Thus, this article presents a brief review of biosensor use in pharmaceutical analysis, focusing on enzymatic electrochemical sensors.