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Enological evaluations capture the chemical and sensory space of wine using different techniques; many sensory methods as well as a variety of analytical chemistry techniques contribute to the amount of information generated. Data fusion, especially integrating data sets, is important when working with complex systems. The success reported when trying to integrate different modalities is generally low and has been attributed to the lack of statistically considerate strategies focusing on the data handling process. Multiple stages of data handling must be carefully considered when dealing with multi-modal data. In this review, the different stages in the data analysis process were examined. The study revealed misconceptions surrounding the process and elucidated rules for purpose-driven approaches by examining the complexities of each stage and the impact the decisions made at each stage have on the resulting models. The two major modeling approaches are either supervised (discrimination, classification, prediction) or unsupervised (exploration). Supervised approaches were emphatic on the pre-processing steps and prioritized increasing performance. Unsupervised approaches were mostly used for preliminary steps. The review found aspects often neglected when it came to the data collection and capturing which in the end contributed to the low success in combining sensory and chemistry data.

The evaluation of the γ-irradiation effects on green teas was holistically achieved by means of a novel algorithm based on linear regression (LRA). This algorithm was compared to the discrimination power of Principal Component Analysis (PCA) and Cluster Analysis (CA). The holistic evaluation was based on positive or negative ion monitoring Electrospray Mass Spectrometry (+/−ESI/MS) data and Reversed Phase Liquid Chromatography with Ultraviolet Spectrometry detection (RPLC/UV) chromatograms, without involving any structural attribution and/or assay of the existing components. Five types of green teas (receiving irradiation doses of 0, 10 and 25 kGy) were considered. Extraction in ethanol and heated water was used. To ensure an increased definition of the profiles being compared, the LRA approach was applied on pairs of large experimental data series resulting from high frequency/high resolution acquisition rates, the resulting slopes, intercepts and correlation coefficients being considered as variables retaining the information contained in the raw data. The discrimination ability varied in the following order: LRA > CA > PCA. The information contained by the input data varied as following: (+)ESI/MS spectra > (-)ESI/MS spectra > RPLC/UV chromatograms.

Dyes and biological sources in 40 samples from red seal threads in 38 documents issued by the Chancery of Moldavia between 1460 and 1503 were investigated by liquid chromatography with UV-Vis (DAD) and mass spectrometric (MS) detection. Lac dye ( Kerria lacca Kerr), redwood type ( Caesalpinia spp.) and madder ( Rubia sp.), as individual dyes or in combinations, were responsible for the colour in all the dyed yarns while tannins were present in more than half of the total number of samples. The presence of major dyes, such as alizarin, purpurin, laccaic acid A and soluble redwood — a marker compound for Caesalpinia species were observed by both DAD and MS detectors while minor compounds (rubiadin, anthragallol, xanthopurpurin, munjistin, flavokermesic acid etc.) were only detected by mass spectrometry. Single stage MS detection was used in the Full Scan mode followed by data processing through Ion Extraction according to the molecular ions of compounds in the database. Tandem MS detection (MS 2 ) was also achieved, through using the Product Ion Scan operating mode. Identification of dyes was made according to retention time, UV-Vis and MS data, based on information collected on standards — dyes and dyed fibers. The biological sources detected are discussed as compared with those identified in ecclesiastical embroideries from the same period, ordered by the same Prince, Stephan the Great (1457–1504).

Styrene induces various adverse health impacts. Due to its wide applications, the quantification of personal exposure level is of great importance, especially in places that attract a large number of people daily, like museums. The aim of this study was to develop a TD-GC-FID based approach to assay the level of residual indoor borne styrene from artifacts consolidated through radio-polymerization. Optimization of the irradiation conditions and the minimum absorbed dose for total radio-polymerization were also investigated through the proposed methodology. Styrene’s assay was performed using a thermal desorption instrument coupled to a gas chromatograph equipped with a flame ionization detector (TD-GC-FID). Specific TD protocols were optimized for both declared objectives. Thermogravimetry/differential scanning calorimetry (TG/DSC) was used for optimization of the TD operational parameters. It was demonstrated that an adsorbed dose of 30 kGy ensures an almost complete radio-polymerization process. Less than 1.746 ppm of styrene is delivered from artifacts and may be inhaled by a person situated in its close proximity, a level placed within the accepted limits imposed by OSHA regulations. It was demonstrated that the TD-GC-FID approach might be useful to assess the occupational exposure to gaseous residual indoor borne styrene from museums exhibiting consolidated artifacts.

A number of ten stable free mono-, di- and tri-radicals of the TEMPO nitroxide type were synthesized and characterized via physico-chemical methods (elemental analysis, MS, UV-Vis, IR, ESR, and X-ray, where appropriate). The design of the compounds was chosen such that supramolecular interaction could be gained via hydrogen-bonding or π–π interactions; therefore the compounds should contain amino- or urea-moieties, (nitro)aromatic rings, or a crown ether residue. The formation of inclusion complexes between these (poly)radicals and cucurbit[6]uril was studied via Electron Spin Resonance (ESR) spectroscopy. The binding constants were evaluated from the analysis of the rotational correlation time dependence on the concentration of cucurbit[6]uril. These constants are an order of magnitude lower than the values reported before for complexes of TEMPO derivatives with β-cyclodextrin. The complexation behaviour of cucurbit[6]uril was revealed in the ESR spectra of the nitroxides in the presence and in the absence of the host molecules, recorded at low temperatures. The experiments revealed that the mobilities of the nitroxides investigated are higher in the absence of the host molecule, a different behaviour compared with complexes with cyclodextrins. This behavior can be understood by taking into account the role played by the presence of salts necessary to ensure the solubility of cucurbit[6]uril in water.

According to some recently published studies, large volume injection (LVI) of diluents immiscible with the eluents used in reversed phase liquid chromatography (RPLC) appears to be feasible, despite the widely accepted rules governing injection related phenomena. The process was previously described and was successfully applied in practice. The present study describes a simple theoretical model explaining LVI of diluents immiscible with the mobile phase in RPLC; the model relies on the on-line coupling of reversed phase supported liquid extraction (RP-SLE) to the chromatographic separation. The compliance of the theoretical model with experimental observations was tested by using data collected for LVI of a homologous series of para-hydroxy alkyl benzoates (methyl, ethyl, propyl, butyl, pentyl, hexyl and octyl congeners) in liquid alkanes (hexane, heptane, iso-octane, decane and dodecane). The SP consisted of an octadecyl chemically modified silica gel eluted with a mixture of acetonitrile:water 4:6 (v/v). Although the model mainly focuses on explaining the linear reduction of the retention time with the injected volume, some aspects relating to zone spreading and thermodynamic aspects are also discussed.

Aim Discrimination power evaluation of UV–Vis and (±) electrospray ionization/mass spectrometric techniques, (ESI-MS) individually considered or coupled as detectors to reversed phase liquid chromatography (RPLC) in the characterization of Ginkgo Biloba standardized extracts, is used in herbal medicines and/or dietary supplements with the help of Fuzzy hierarchical clustering (FHC). Experimental Seventeen batches of Ginkgo Biloba commercially available standardized extracts from seven manufacturers were measured during experiments. All extracts were within the criteria of the official monograph dedicated to dried refined and quantified Ginkgo extracts, in the European Pharmacopoeia. UV–Vis and (±) ESI-MS spectra of the bulk standardized extracts in methanol were acquired. Additionally, an RPLC separation based on a simple gradient elution profile was applied to the standardized extracts. Detection was made through monitoring UV absorption at 220 nm wavelength or the total ion current (TIC) produced through (±) ESI-MS analysis. FHC was applied to raw, centered and scaled data sets, for evaluating the discrimination power of the method with respect to the origins of the extracts and to the batch to batch variability. Results The discrimination power increases with the increase of the intrinsic selectivity of the spectral technique being used: UV–Vis

The altered profiles of prolactin secretion in the anterior hypophysis, generated by pathological, pharmacological or toxicological causes, have special consequences on multiple functions in both genders.This selective review presents the main mechanisms controlling prolactin secretion, focusing on the interplay of various neurotransmitters or xenobiotics, but also on the role of psychic or posttraumatic stress. A detailed analysis of several pharmacotherapeutic groups with hyperprolactinemic effects emphasize on the relevance of the pharmacokinetic/pharmacodynamic mechanisms and the clinical significance of the long term administration.Accurate monitoring and evaluation of the hyperprolactinemia induced by xenobiotics is strongly recommended. The typical antipsychotics and some of the atypical agents (amisulpride, risperidone, paliperidone), as well as some antidepressants, antihypertensives and prokinetics, are the most important groups inducing hyperprolactinemia. The hyperprolactinemic effects are correlated with their affinity for dopamine D2 receptors, their blood-brain barrier penetration and, implicitly, the requested dose for adequate occupancy of cerebral D2 receptors. Consequently, integration of available pharmacokinetic and pharmacodynamic data supports the idea of therapeutic switch to non-hyperprolactinemic agents (especially aripiprazole) or their association, for an optimal management of antipsychotic-induced hyperprolactinemia. Possible alternative strategies for counteracting the xenobiotics-induced hyperprolactinemia are also mentioned.

Diode array spectrometry covers a broad range of wavelengths, from soft X-rays to the near-infrared (NIR) domain. Complete spectra can be obtained in

A pulsed dielectric barrier discharge was used for the decomposition of enalapril, its metabolite, enalaprilat, its degradation by-product, diketopiperazine, aswellastheircombination, in aqueous solution. The discharge was generated at the interface between gas and the solution which flows as a film on the surface of the inner electrode of the plasma reactor. The degradation mechanism of the studied compounds and the temporal evolution of the reaction intermediates were established from LC‐MS analysis. A wide variety of degradation by-products were formed; at least six new derivatives, not mentioned in literature, were observed. However, most of these by-products were subsequently degraded to small molecules. The mineralization degree, determined from TOC and COD measurements, was approximately 40%.

Dimethyl sulfate (DMS) is frequently used in pharmaceutical manufacturing processes as an alkylating agent. Trace levels of DMS in drug substances should be carefully monitored since the compound can become an impurity which is genotoxic in nature. Derivatization of DMS with dibenzazepine leads to formation of the N-methyl derivative, which can be retained on a reversed phase column and subsequently separated from other potential impurities. Such derivatization occurs relatively slowly. However, it can be substantially speed up if ionic liquids are used as reaction media. In this paper we report the use of 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide (IL1) and 1-butyl-4-methylpyridinium tetrafluoroborate (IL2) as reaction media for the derivatization of DMS with dibenzazepine. It was determined that the stoichiometry between the substrate and DMS may be 1:1 or 2:1, in relation with the nature of the reaction media. An (+)ESI-MS/MS approach was used for quantitation of the derivatized product. Alternatively, DMS derivatization may be carried out with pyridine in acetonitrile (ACN). The N-methylpyridinium derivative was separated by hydrophilic interaction liquid chromatography (HILIC) and detected through (+)ESI-MS (in the SIM mode). In both cases, a limit of quantitation (LOQ) of 0.05 μg/ml DMS was achievable, with a linearity range up to 10 μg/ml. Both analytical alternatives were applied to assay DMS in 4-(2-methoxyethyl)phenol, which is used as a starting material in the synthesis of metoprolol.

The absorption, distribution, metabolism, excretion and toxicity (ADME(T)) of oxime reactivators have been assessed with respect to their polarity, a fundamental requirement for their specific mechanism of action in the intoxication with organophosphorous compounds. The limitations of the therapeutic outcome have been associated not only with the severity of intoxication and to particularities of the toxicants, but also to the reduced lipophilicity and consequent restricted permeability across biological barriers.This article inventories the plethora of mnemotic rules developed throughout the years for defining chemical spaces where drugs share one or more structural and ADME(T) characteristics. Their applicability to oxime is analyzed, especially in relation to intestinal absorption and brain distribution. Other aspects of oximes for antidotal outcome are also reviewed.The drugability rules are not applicable to oxime reactivators, because the increase in lipophicity and consequent improved permeability across biological barrier comes together with amplified (neuro)toxicity and reduced reactivating capacity. The available data suggest a high solubility and reduced metabolism, assigning the quaternary oximes to the fourth class of Biopharmaceutical Classification Systems. Reliance upon oral absorption data for designing safe centrally acting oximes can be of potential value, with adequate characterization of uptake-influx transporters interplay.

Lignin isolated from Miscanthus × giganteus using acidic (FAL) and basic (AL) conditions was thereafter subjected to catalytic depolymerization under thermal or ultrasonic activation. The characterization of lignin samples was achieved by thermogravimetric analysis and FTIR. Three different classes of catalysts containing nickel as the active species have been prepared in this respect: (i) nano-Ni(0) by reduction of NiCl 2 with NaBH 4 under ultrasonication, (ii) Fe 3 O 4 –(NiMgAlO) x and (NiAlO) x by calcination of Mg(Ni)–Al hydrotalcite incorporating Fe 3 O 4 , followed by reduction with hydrogen, and (iii) NiO(1 1 1) nanosheets by reduction of Ni(NO 3 ) 2 with urea using benzyl alcohol as a structure directing agent. The catalysts were characterized by XRD and XPS techniques. Reduced mixed oxides displayed a moderate activity while a significant increase in conversion (up to 90%) was observed with nano-Ni(0) and NiO(1 1 1) nanosheets catalysts. The conversion and the mass distribution of the reaction products were strongly related to the procedure used for the extraction of lignin. In all the reactions AL samples had a greater extent of depolymerization. The performance from the tested catalysts under ultrasonic conditions was inferior to those tested under conventional heating conditions. The nature of the solvent was also found to be very important to this process, with the ionic liquid, 1-butyl-3-methylimidazolium acetate ([BMIM]OAc) demonstrating the best results in autoclave conditions and methanol under ultrasonic conditions.

Injectable solutions used in treatment of intense pain are based on combinations of active ingredients such as metamizole sodium (MTZ), pitofenone hydrochloride (PTF) and fenpiverine bromide (FPB). The simultaneous chromatographic assay of such combinations poses difficulties due to their structural variety, highly polar character, and wide concentration ranges (500 mg mL−1 for MTZ, 2 mg mL−1 for PTF and 0.02 mg mL−1 for FPB). Fast hydrolysis of MTZ on aqueous dilution causes additional problems due to impurity (MTC) formation. Sodium hexane sulphonate (10 mM) was used as ion pairing agent for PTF, FPB and MTC in a mobile phase consisting of 48/52 (v/v) methanol and aqueous 0.2% triethylamine at pH=3. The ionic liquid 1-butyl-1-methyl-pyrrolidinium tetrafluoroborate (10 mM) was used as mobile phase additive to preserve the MTZ peak symmetry. The minor active ingredient FPB was selectively extracted into 1-octanol by ion pair formation with picric acid. A 20 µL aliquot of the organic layer was directly injected into the column.

The metabolic processes frequently trigger highly complex pharmacokinetic (PK) and pharmacodynamic (PD) characteristics for the coexisting entities, parent drug and its active or inactive metabolites. The interpretation of both individual and cumulative profiles, frequently used in the therapeutic drug monitoring procedures, must take into consideration the biological coherence of the changes of the molecular descriptors characterizing the metabolites versus the parent drugs, and further qualitative and quantitative consequences on permeability processes across highly specialized biological barriers (e.g. blood–brain barrier [BBB]). This paper analyzes the correlation of molecular descriptor differences and the PK/PD consequences for three representative psychotropic drugs (risperidone, clozapine and tramadol) and their active metabolites, underlying the safety and efficacy concerns of using the products of metabolic processes as potential new drugs. The minimal structural changes are correlated with the predicted or experimental penetrability across the biological membranes, with a special emphasis on BBB penetration, as the limiting phase for the effect at central nervous system level. The PD characteristics related to the active metabolites are compared to the ones reported for the parent drugs, concerning mainly the affinity for cerebral receptors and the type of activity at a specific level. For the neuropsychotropic substances, with BBB penetrability as a sine qua non condition, the comparative analysis of PK/PD properties for the parent drug and its metabolites generates a complete and highly complex image of the consequences of their coexistence, since these entities must be conceived and analyzed not separately, but by inclusion of usually complementary properties generating a unique therapeutic profile.

Limonene, considered a green solvent, was successfully used to extract simvastatin, lovastatin, and their hydroxy-acid metabolites from human plasma samples. The extraction process was followed by the direct injection of a large volume aliquot (100 μL) from the limonene layer into a Zorbax SB-C18 Rapid Resolution chromatographic column (50 mm length × 4.6 mm i.d. × 1.8 µm d.p.), operated under gradient elution reversed-phase separation mechanism. Tandem mass spectrometry operated under the multiple reaction monitoring mode was used for detection, providing low quantitation limits in the 0.25–0.5 ng/mL concentration interval. This method was validated and used for quantitation of simvastatin and its hydroxy acid metabolite in incurred plasma samples obtained from two volunteers participating in a bioequivalence study, using lovastatin and its hydroxy analog as internal standards. The results were statistically compared with those produced by means of an alternative RPLC-tandem MS using protein precipitation with acetonitrile. The quality attributes of the two methods are comparatively discussed. The agreement between the quality characteristics of the two methods and the experimental results obtained on real samples may be considered as a consistent basis for the simultaneous use of limonene as extraction medium and injection diluent for hydrophobic compounds in bioanalytical approaches. Copyright © 2012 John Wiley & Sons, Ltd.

The bioequivalence of two formulations of 15 mg tablets of meloxicam (CAS 71125-38-7), Meloxicam (LaborMed Pharma) and a commercially available preparation as reference in 24 healthy male and female Caucasian volunteers was assessed based on a validated non-extractive HPLC-DAD method. The sample preparation procedure was based on protein precipitation with a mixture of methanol/acetonitrile, trifluoacetic acid and sodium sulfate solution. Piroxicam (CAS 36322-90-4) was used as internal standard. The stepwise gradient elution profile of the chromatographic method allows injection of a high volume of the sample (500 microL) with the focusing of both analytes in a Chromolith Performance RP-18e column. The mobile phase constituents are methanol and aqueous 20 mmol/L Na2HPO4 buffer solution brought to pH = 6 with H3PO4. A flow-rate of 2 mL/min achieves a complete chromatographic run (including column equilibration) within 12 min. UV detection at 356 nm allowed a quantitation limit around 30 ng/mL. Bioequivalence study was based on an open-label, randomized, two-period, two-sequence, single dose, crossover design with a 2-week wash-out period between consecutive oral administrations. The main pharmacokinetic parameters (C(max), t(max), t 1/2, AUD and AUC(0-infinity)) were considered as evaluation criteria for the bioequivalence of the test drug against the reference.

Background: Liquid–liquid extraction of target compounds from biological matrices followed by the injection of a large volume from the organic layer into the chromatographic column operated under reversed-phase (RP) conditions would successfully combine the selectivity and the straightforward character of the procedure in order to enhance sensitivity, compared with the usual approach of involving solvent evaporation and residue re-dissolution. Large-volume injection of samples in diluents that are not miscible with the mobile phase was recently introduced in chromatographic practice. The risk of random errors produced during the manipulation of samples is also substantially reduced. Results: A bioanalytical method designed for the bioequivalence of fenspiride containing pharmaceutical formulations was based on a sample preparation procedure involving extraction of the target analyte and the internal standard (trimetazidine) from alkalinized plasma samples in 1-octanol. A volume of 75 µl from the octanol layer was directly injected on a Zorbax SB C18 Rapid Resolution, 50 mm length × 4.6 mm internal diameter × 1.8 µm particle size column, with the RP separation being carried out under gradient elution conditions. Detection was made through positive ESI and MS/MS. Aspects related to method development and validation are discussed. Conclusions: The bioanalytical method was successfully applied to assess bioequivalence of a modified release pharmaceutical formulation containing 80 mg fenspiride hydrochloride during two different studies carried out as single-dose administration under fasting and fed conditions (four arms), and multiple doses administration, respectively. The quality attributes assigned to the bioanalytical method, as resulting from its application to the bioequivalence studies, are highlighted and fully demonstrate that sample preparation based on large-volume injection of immiscible diluents has an increased potential for application in bioanalysis.

Background: The inherent reproducibility of a bioanalytical approach is usually sustained through incurred sample reanalysis (ISR). Questions relating to the number of ISRs, the right moment for performing reanalysis, the way of performing an appropriate statistical refinement of experimental data and actions to be taken in the case of failure are frequently raised. Results: Data resulting from ISR following a bioequivalence study for spironolactone formulations are discussed. Reanalysis of samples was carried out twice: immediately after the end of the study and after a period that overcame the long-term stability study achieved during method validation. The Bland–Altman approach was used to assess experimental results. ISR was successful over the short reanalysis period for both compounds. Data produced through reanalysis after the long-term period indicated a systematic positive bias for the metabolite canrenone (although results supported reproducibility). The results obtained for spironolactone were affected by a strong negative systematic bias and failed to support reproducibility. The explanation deals with the continuous conversion of spironolactone to canrenone in plasma samples. However, reproducibility of the method may be sustained by comparing original and repeated differences between concentration values in samples by means of a paired t-test, Wilcoxon sign rank-sum test and linear regression. Conclusions: Different statistical approaches for making data comparisons are discussed and may be successfully applied during reanalysis of samples from a bioequivalence study. Results of the evaluations may differ in accordance with the statistical procedure being applied, thus a definitive conclusion requires consideration of all specific experimental circumstances arising during production of the processed data.

Calibration data of LC-MS/MS rarely fit the pure least square regression model, especially for large concentration intervals. The response function of the MS instrument is corrected by weighted regression models or logarithms. The choice of a response linearization method is based on results produced through back-interpolation of experimental data and/or evaluation of correlation coefficients. Two bioequivalence studies carried out for pharmaceutical formulations containing metformin gave us the opportunity to appreciate the impact of the MS response linearization method (logarithm and 1/x weighted linear regression) on method quality characteristics. The sample preparation was based on protein precipitation with acetonitrile. Chromatographic separation was achieved on a Zorbax CN column (mobile phase acetonitrile and aqueous 10 m m ammonium acetate solution, pH 3.5). Tandem MS detection was performed on a triple quadrupole spectrometer equipped with an electrospray source, operated under positive-ion mode. The method was validated and used for evaluation of the bioequivalence of formulations containing 500 and 1000 mg metformin. The 500 mg metformin study used logarithms for linearization of the detector response, while the 1000 mg metformin study was based on 1/x linear weighted regression. Data resulting from validations and studies completion were compared with evaluate the impact of the response linearization on the method quality characteristics.

Large volume injection of samples in strong diluents immiscible with the mobile phases used in reversed phase liquid chromatography (RPLC) has been recently introduced in practice. In the present work, the potential of the technique has been evaluated for bioanalytical applications. The process consists of the liquid-liquid extraction of indapamide from whole blood into 1-octanol, followed by the direct injection from the organic layer into the LC. Detection was made through negative electrospray ionization (ESI) and tandem mass spectrometry (MS(2)). The method was developed, validated, and successfully applied to a large number of samples in two bioequivalence studies designed for indapamide 1.5mg sustained release and 2.5mg immediate release pharmaceutical formulations. The performance of the analytical method is discussed based on data resulting from the validation procedure and the completion of the bioequivalence studies.

Thermodynamic parameters, such as standard enthalpy (ΔH0) and entropic contributions (ΔS0/R + volume phase ratio) of the partition between mobile and stationary phases, for several polar model compounds of pharmaceutical interest, were estimated in RP-LC for different flow-rates and using an octadecyl-silicagel stationary phase. The temperature range used to plot van't Hoff curves was 25–50°C, which is the usual range for separation in RP-LC. Below 20°C, van't Hoff plots become non-linear for all studied compounds, due to the changes of the balance between hydrophobic and solvophobic forces that influence the retention process. Other significant deviations were observed for one compound that is involved in inter-changeable tautomeric equilibria (as discussed for piroxicam). Regression parameters of the linear dependences obtained between retention (natural logarithm of retention factor, ln k') and reciprocal absolute temperature (1/T) together with the estimated phase ratio were used to calculate the ther...

The ESI–MS and MS/MS behavior of functionalized calix[4]arenes (1–5) has been studied in both positive and negative-ion mode. Liquid chromatography coupled to ESI–MS has been successfully used for separation of the byproducts issuing from the functionalization pathways, through the application of a simple reversed phase mechanism. The ability of (1–5) to host methyl esters of amino acids, tyrosine, tryptophan, phenylalanine, cysteine, valine, serine, leucine, isoleucine, and threonine has been evaluated by means of MS identification of the host–guest resulting in protonated molecular ions. The direct infusion within the ESI source of the solutions containing the two partners (i.e., calixarene and amino acid derivative) could act as a fast screening means for the evaluation of hosting capability. Only positive ionization may offer information about the host–guest complexes being formed. The influence of the excess of a partner in the infused solution strongly alters ionization yields, making quantitative approaches meaningless. Attempts to chromatographically isolate the host–guest complexes failed, probably due to the fact that interactions of the partners with the mobile and stationary phases are higher than the inclusion interactions. Structures consisting of combined fragments of the host–guest partners resulting from the collisional induced dissociation have not been observed.

A new sensitive HPLC/MS/MS method for simultaneous determination of furosemide, spironolactone and canrenone in human plasma samples is presented. Electrospray ionization source (ESI) has been used. The tandem MS detection was performed under MRM conditions, in the negative ion mode for furosemide and indapamide (internal standard 1) and in the positive ion mode for spironolactone, canrenone and nitrazepam (internal standard 2). A simple plasma protein precipitation with acetonitrile was used as sample preparation technique. The chromatographic separation was carried out under the reversed phase mechanism, on a 250 mm length column packed with octadecyl modified silicagel and thermostated at 35 degrees C. The column was operated under isocratic conditions (3:7 aqueous 0.1% formic acid and methanol, v/v) at a flow rate of 0.8 mL/min. Quantitation intervals of 20-1600 ng/mL for furosemide and 2-100 ng/mL for spironolactone and canrenone have been concluded through validation. Precision and accuracy were situated within the accepted thresholds (maximum 15% relative standard deviation and +/-15% percentage bias). The most sensitive aspects relating to the analytical method development and validation were highlighted and critically assessed in order to reach an objective opinion about the real performances and inherent applicability of the method in bioanalysis.

Chromatographic retention data obtained from interactions between some oxime-type compounds and different stationary phases (involving hydrophobic interaction, ion pairing formation availability, pi-pi, H-bonding, dipole-dipole, ion-dipole, electrostatic interaction and glycoprotein binding ability) have been studied. The logarithms of the capacity factors extrapolated at 0% or 100% organic solvent, resulting from the functional dependencies between retention and mobile phase composition, were used for estimation of different kind of hydrophobicity or hydrophilicity descriptors (HHDs) of these compounds. The conditions of the chromatographic separation were chosen as close as possible to in-vivo conditions (the aqueous component of the mobile phase has a pH in the physiologic interval 6.8-7.2, 0.9% sodium chloride was added to reproduce ionic strength and isotonic character, and the temperature was set at 37 degrees C). These descriptors characterizing the partition between stationary/mobile phases through specific interactions may be directly used for correlation to biological distribution processes, such as penetration of the blood/brain barrier. Oxime-type compounds used as acetylcholinesterase (AChE, E.C.3.1.1.7) reactivators have been considered for the retention study. The choice is supported by their use in the therapy of acute intoxication with organophosphorus AChE inhibitors (OPIs, especially nerve agents and pesticides), a rather complicated chemistry in solution and a relative lack of data about computational molecular descriptors used for modeling biological partition/distribution. Some correlations between the determined descriptors and computational values have also been discussed.

Undoubtedly, the use of oximes represents real progress in counteracting intoxications with organophosphates (OP), through potentiating antidotal effects of atropine. The penetration extent of these compounds through the blood-brain barrier (BBB) to significantly reactivate phosphorylated or phosphonylated acetylcholinesterase (AChE) in the brain still remains a debatable issue. Penetration of biological barriers by oximes was investigated mainly through determination of several quantitative parameters characterizing digestive absorption and BBB penetration. A weak penetration of biological barriers could be concluded from the available experimental data. The functional parameters/therapeutic effects following the penetration of oximes through BBB, more precisely the antagonism of OP-induced seizures and hypothermia, prevention of brain damage and respiratory center protection, leading to the final end-point, the survival of intoxicated organisms, are of high interest. It seems obvious that oximes are weakly penetrating the BBB, with minimal brain AChE reactivation (

Hyphenation of mass spectrometry (MS) to liquid chromatography (LC) represents a powerful tool for qualitative and quantitative characterization of target compounds in very complex matrixes of biological origins. In spite of many advantages due to recent advances and innovations in the area of instrumentation and dedicated software support, some difficulties are still encountered in its current applications. The large variety of functional principles and technical solutions applied for hyphenation of the two techniques, for ion sources, ion extraction and focusing, mass analysis, and ion counting makes it more difficult to obtain perfect agreement between the intrinsic characteristics of the laboratory-available instrumentation and the declared goals of specific determination. This review covers a part of the literature data dealing with the shortcomings of LC/MS in bioanalysis. The following topics are discussed: structural identification and confirmation in LC/MS; precision of the instrumental response ...

Identification of dyes in historic textiles was until recently only based on reversed phase liquid chromatography and diode-array detection (RPLC–DAD). Although in the last years mass spectrometry (MS) is increasingly used as a detection system for liquid chromatography, most applications in the field are directed to identification of the molecular ions or in studies dedicated to degradation products which may be used as markers in RPLC–DAD. In the present work, an analytical protocol for the identification of dyes using RPLC/ESI/MS is presented. Atmospheric pressure electrospray ionization (ESI) was applied, in the negative ion monitoring mode. Both single stage and tandem MS (MS/MS) approaches were considered. An ion trap was used as mass analyzer. Experiments are based on the characterization of standards (natural dyes and/or dyed fibers) with the mass spectrometer sequentially working in the following modes: single MS/full scan, followed by plotting chromatograms through ion extraction (IEC) according to mass/charge ratios corresponding to molecular ions; single MS/selected ion monitoring (SIM) mode; tandem MS/single reaction monitoring (SRM) mode; tandem MS/multiple reactions monitoring (MRM) or product ion scanning modes. A faster chromatographic separation could be applied as MS detection readily balanced the selectivity of the analytical process. In a case study, 11 dyes from 3 biological sources were detected in a 0.5 mg historic sample.

Assay of cyclohexanamine as related impurity in glipizide (as active ingredient or in its pharmaceutical formulations) has been achieved through pre-column derivatization with o-phtaldialdehyde (OPA) followed by the HPLC/DAD assay. Derivatization reaction was found to be fast, quantitative, and produces required sensitivity under UV detection at 245 nm. The absolute limit of quantitation is placed around 0.05 μg/mL, meaning less than 0.001% with respect to the amount of glipizide used in the test solution and 1/10 from the maximum threshold imposed for cyclohexanamine in glipizide through the European Pharmacopoeia compendial monograph. No interferences from the derivatization reagent or its related by-products and from the other impurities of glipizide were observed. The assay was validated according to guidances in force. The precision of the method is placed within the 10% interval (expressed as relative standard deviation) while accuracy is placed in the ±10% range (expressed as % bias). This method c...

Retention of some cationic-type aldoximes used as AChE reactivators was studied under ion-pairing (IP) liquid chromatographic conditions, using alkyl sulphonates as IP agents. An unusual chromatographic retention behavior was observed: the functional dependencies between the logarithm of the capacity factor and the methanol content in the mobile phase followed a binomial pattern (U-shaped), with a minimum positioned within the interval 50–80% methanol. The tautomerism of the investigated structures and the stationary phase morphological modification caused by the adsorption of the ion-pairing agent may explain the experimental findings. The study focused on the influences brought by the pH, the alkyl chain length of the ion-pair agent, and the organic modifiers on the retention behavior of these compounds.

Direct plasma loading on a LiChrospher ADS C18 cartridge on-line coupled to an analytical Zorbax XDB C18 column was used to analyze felodipine by means of positive atmospheric pressure chemical ionization (APCI) tandem mass spectrometric (MS/MS) detection. Appropriate sensitivity, accuracy, and precision were obtained using an ion trap mass analyzer operated in a multiple reaction monitoring (MRM) mode. Diethyl-4-(2,3-dichlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate) was used as an internal standard. The method was fully validated. The method was successfully applied to a pilot bioequivalence study made on extended release oral dosage forms containing 10 mg of felodipine, under fed and fasting conditions.

The pharmacokinetic characteristics of a compound as well as the immediate consequences of its physicochemical behavior during interactions with biological structures, represent the key issues for its pharmacodynamic profile, starting from the most fundamental global aspects (i.e. central and / or peripheral action) to the most detailed ones (i.e. molecular mechanism of action). Successive metabolic reactions lead to either bioactivation or bioinactivation of the molecular entity. A particular importance is currently assigned to several molecular physicochemical descriptors, for instance the polarity degree (mirroring the changes of partition coefficients and of the permeability of biological structures), and emphasizing on distribution and renal excretion rate. The active metabolite (9-hydroxy-risperidone) of the atypical antipsychotic agent risperidone has an increased polar character and, consequently, its pharmacokinetic profile is modified compared to the parent drug: especially the penetration through the bood brain barrier and the efflux pump mediated transport were considered. In this context, the kinetic characteristics and their correlation with the pharmacodynamic properties for the two active entities, as well as the consequences dealing with the antipsychotic efficacy, the safety and efficacy profiles can be anticipated. The present approach critically asseses the available data from literature corroborated with the personal findings over the last years.

The sensitivity of the compendial European Pharmacopoeia RPLC/UV method for assaying residual ginkgolic acids in Ginkgo biloba standardized extracts was essentially improved by operating modifications on the sample preparation procedure, including sample injection. The acidified methanol/water solution of the Ginkgo biloba standardized extract was extracted “in situ” with n-hexane (concentration factor of 20). From the organic layer, a relatively large volume (50 µL) of the hexane solution was directly loaded to the chromatographic column. A modified gradient elution profile was used in order to force the sample solvent to elute faster than analytes, without affecting peak symmetry. The separation is obtained in 22 minutes. As the amount of the target compounds loaded to column is higher, selective UV detection at 310 nm was possible. The method was validated according to specific operating guidelines for selectivity, linearity range, quantitation limits, precision, accuracy, and robustness. A limit of qu...

Assay of ethyl-4-oxopiperidine-1-carboxylate (Impurity H) as related impurity in loratadine (active ingredient or in its pharmaceutical formulations) has been achieved through precolumn derivatization with 2,4-dinitrophenylhydrazine followed by HPLC/DAD. The derivatization reaction was found to be selective, fast, quantitative, and allows good sensitivity when monitored at 368 nm detection wavelength (absolute limit of quantitation is placed around 0.1 µg/mL, meaning 0.01% with respect to the amount of loratadine used in the test solution and 1/10 from the maximum allowed threshold for impurity H). Derivatization conditions and kinetics of the reaction are discussed. For convenience, the HPLC method used for separation of the derivatization product is identical to the method described in the European Pharmacopoeia 6.0, loratadine monograph, the test for related substances (other than impurity H). No interferences from the other compendial cited impurities of loratadine and from the derivatization...

A sensitive method for determination of free captopril as monobromobimane derivative in plasma samples is discussed. The internal standard (IS) was 5-methoxy-1H-benzimidazole-2-thiol. Derivatization with monobromobimane immediately after blood collection and plasma preparation prevents oxidation of captopril to the corresponding disulfide compound and enhances the ionization yield. Consequently, derivatization enhances sample stability and detection sensitivity. Addition of the internal standard was made immediately after plasma preparation. The internal standard was also derivatized by monobromobimane, as it contains a thiol functional group. Preparation of plasma samples containing captopril and IS derivatives was based upon protein precipitation through addition of acetonitrile, in a volumetric ratio 1:2. The reversed-phase liquid chromatographic separation was achieved on a rapid resolution cartridge Zorbax SB-C(18), monitored through positive electrospray ionization and tandem MS detection using the multiple-reaction monitoring mode. Transitions were 408-362 amu for the captopril derivative and 371-260 amu for the internal standard derivative. The kinetics of captopril oxidation to the corresponding disulfide compound in plasma matrix was also studied using the proposed method. A linear log-log calibration was obtained over the concentration interval 2.5-750 ng/mL. A low limit of quantitation in the 2.5 ng/mL range was obtained. The analytical method was fully validated and successfully applied in a three-way, three-period, single-dose (50 mg), block-randomized bioequivalence study for two pharmaceutical formulations (captopril LPH 25 and 50 mg) against the comparator Capoten 50 mg.

A chromatographic column packed with 1.8 microm particle size octadecyl modified silicagel was used to separate terpene trilactones from Ginkgo biloba extracts/pharmaceutical formulations. Gradient elution was applied, using acidic methanol and water as mobile phase components (0.1% formic acid addition). No specific sample preparation is needed, except dissolution/extraction in methanol of the solid material. Baseline separation of bilobalide and ginkgolides A, B, C and J is obtained within 4 min. The gradient profile is needed to elute the remaining matrix from column. A separation cycle takes 7 min, including column re-equilibration. MS/MS detection with positive electrospray ionization and triple quadrupole mass analysis was used. Multiple reaction monitoring mode was applied for data acquisition, taking protonated molecular ions as precursors. In order to generate reproducible ionization conditions, the standard addition method was considered. The assay of terpene trilactones obtained under such conditions was validated according to guidances in place. Method intermediate reproducibility corresponds to a relative standard deviation of less than 10%. Accuracy, expressed in terms of absolute percent bias ranged from 90% to 110%. Spectral confirmation of target analytes was also included in the validation procedure.

A bioequivalence study of two formulations containing norfloxacin was used for identification and assay of the metabolite of norfloxacin in human serum samples. The bioequivalence study was based on an analytical method using liquid chromatography with fluorescence detection. The plasmatic profile of metabolite was similar to norfloxacin for both formulations. Three plasma fractions of norfloxacin metabolite from a volunteer were isolated by liquid chromatography and investigated by atmospheric-pressure chemical ionization mass-spectrometry. The structure of norfloxacin metabolite (7-aminoethylenamino-6-fluoro-4-hydroxy quinoline-3-carboxylic acid) was identified taking into account also the mass spectrometry investigations achieved for norfloxacin and ciprofloxacin (used as internal standard for the analytical method). A theoretical procedure based on quantum chemical calculations has been also used to explain the mass fragmentation in molecules of norfloxacin metabolite that differ from the molecule of norfloxacin.

Injection of large volumes of samples in solvents other than mobile phase composition has been proved for some less hydrophobic compounds. Thus, the retention behavior of several compounds of pharmaceutical interest (isosorbide-2-nitrate, isosorbide-5-nitrate, tropicamide, pentoxifylline, and methyl p-hydroxybenzoate) was studied by using different hydrophobic solvents (n-hexane, n-heptane, or i-octane) as sample solvents. Two types of stationary phases were used: octyl and octadecyl modified silica (both of Zorbax Eclipse type). The experiments showed a linear dependence between capacity factor of each solute and sample injection volume, up to maximum volume values of about 680 microL for C8 stationary phase and 580 microL for C18 stationary phase, when the solutes are no longer retained in stationary phase. Injection of large volumes of these hydrophobic solvents is thus possible in RP-LC with a gradual reduction of retention and peak efficiency. Two major conditions are however necessary in order to apply such an injection approach: the solutes must have a proper solubility in hydrophobic solvents and meanwhile they have to be less hydrophobic than the sample solvent in order to avoid competition with solvent molecules in partitioning between mobile and stationary phases.

Chromatographic retention of some phenolic compounds (impurities of related pharmaceutical active substances) was studied for different organic modifier contents in mobile phase. Their capacity factors k′ versus organic modifier content (acetonitrile, or methanol) in mobile phase were measured and the corresponding dependences were studied by linear and polynomial regressions and then they were extrapolated to a mobile phase with no content in organic modifier in order to estimate their octanol/water partition coefficient (log Kow). The values of log Kow from retention data were compared to known shake‐flask experimental values and theoretical values predicted by fragment methodology. Linear dependences for methanol and second‐degree polynomial dependences for acetonitrile between organic modifier volume percentage and log k′ provided good estimation of log Kow. The chromatographic method can be considered as a method of choice for estimating hydrophobicity parameter for different solutes. The l...

The influence of the experimental variability (instrumental repeatability, instrumental intermediate precision and sample preparation variability) and data pre-processing (normalization, peak alignment, background subtraction) on the discrimination power of multivariate data analysis methods (Principal Component Analysis -PCA- and Cluster Analysis -CA-) as well as a new algorithm based on linear regression was studied. Data used in the study were obtained through positive or negative ion monitoring electrospray mass spectrometry (+/-ESI/MS) and reversed phase liquid chromatography/UV spectrometric detection (RPLC/UV) applied to green tea extracts. Extractions in ethanol and heated water infusion were used as sample preparation procedures. The multivariate methods were directly applied to mass spectra and chromatograms, involving strictly a holistic comparison of shapes, without assignment of any structural identity to compounds. An alternative data interpretation based on linear regression analysis mutually applied to data series is also discussed. Slopes, intercepts and correlation coefficients produced by the linear regression analysis applied on pairs of very large experimental data series successfully retain information resulting from high frequency instrumental acquisition rates, obviously better defining the profiles being compared. Consequently, each type of sample or comparison between samples produces in the Cartesian space an ellipsoidal volume defined by the normal variation intervals of the slope, intercept and correlation coefficient. Distances between volumes graphically illustrates (dis)similarities between compared data. The instrumental intermediate precision had the major effect on the discrimination power of the multivariate data analysis methods. Mass spectra produced through ionization from liquid state in atmospheric pressure conditions of bulk complex mixtures resulting from extracted materials of natural origins provided an excellent data basis for multivariate analysis methods, equivalent to data resulting from chromatographic separations. The alternative evaluation of very large data series based on linear regression analysis produced information equivalent to results obtained through application of PCA an CA.

Bromazepam was isolated from plasma samples by simple protein precipitation with acetonitrile. A high throughput isocratic reversed phase separation between bromazepam and nitrazepam (IS) was achieved within 2 minutes on a rapid resolution cartridge column Zorbax SB‐C18, using methanol/aqueous 0.1% formic acid 40/60 (v/v) as mobile phase. Detection was made by tandem mass spectrometry, using the multiple reaction monitoring mode (MRM). Both electrospray (ESI) and atmospheric pressure chemical (API) ionizations modes were considered, together with triple quadrupole (TQ) and ion trap (IT) mass analyzers. Ionization patterns are discussed; no major differences being observed between ESI and API. Higher sensitivity was obtained when using the TQ mass analyzer. Comparison between different ionization modes/mass analyzers types is made, based on bromazepam concentration/time profiles in real plasma samples. The best results were obtained in ESI/TQ conditions. Consequently, the method was fully validate...