Combination of large volume sample stacking with polarity switching and cyclodextrin electrokinetic chromatography (LVSS-PS-CDEKC) for the determination of selected preservatives in pharmaceuticals.

In this study, a large volume sample stacking (LVSS) with polarity switching (PS) and cyclodextrin electrokinetic chromatography (CDEKC) method has been developed for the simultaneous separation and determination of 8 preservatives: methylparaben (MP), ethylparaben (EP), propylparaben (PP), butylparaben (BP), isobutylparaben (IBP), sorbic acid (SA), benzoic acid (BA), p-hydroxybenzoic acid (PHBA) in pharmaceuticals. The effects of some typical parameters such as sample volume, applied voltage, composition and pH of the running buffer and organic modifier concentration were examined and optimized. Moreover, the impact of type and concentration of cyclodextrin as electrolyte modifiers was also investigated. The detection limits of analytes for the elaborated LVSS-PS-CDEKC method were found to be in 0.8-5 ng mL-1 range, which were around 500 times lower than normal CDEKC without preconcentration technique. All analytes were completely resolved in less than 11 min in an uncoated fused-silica capillary of 75 µm internal diameter (I.D) x 50 cm length. The electrophoretic separation was performed in a 2 mM α-cyclodextrin and 25 mM tetraborate system (pH = 9.3) with an applied voltage of 25 kV. The established method was validated and confirmed to be applicable for the determination of the preservatives in a quality control of pharmaceuticals.

Pharmaceutical compounding of orphan active ingredients in Belgium: how community and hospital pharmacists can address the needs of patients with rare diseases.

BACKGROUND: Pharmaceutical compounding of orphan active ingredients can offer cost-effective treatment to patients when no other drug product is available for a rare disease or during periods of drug product shortages. Additionally, it allows customized therapy for patients with rare diseases. However, standardized compounding formulas and procedures, and monographs are required to ensure the patients' safety. RESULTS: Standardized formulas and compounding procedures were developed for seven orphan active ingredients (L-arginine, sodium benzoate, sodium phenylbutyrate, L-carnitine, chenodesoxycholic acid, primaquine phosphate, pyridoxal phosphate) and one non-orphan molecule (sodium perchlorate) regularly compounded by hospital pharmacists for extemporaneous use. The stability of these formulations was evaluated over 3 months at refrigerated (5 °C) and standard storage conditions (25 °C/60%RH) using HPLC-based assays and a suitable shelf life was assigned to the formulations. Additionally, suitable analytical methods for quality control of formulations of pyridoxal phosphate and sodium perchlorate were developed as monographs for these components were not available in the European Pharmacopeia or United States Pharmacopeia. CONCLUSIONS: Availability of compounding formulas and protocols, as well as stability information, for orphan active ingredients can improve patients' access to treatment for rare diseases. Such data were collected for seven orphan active ingredients to treat patients with rare diseases when no other treatment is available. More efforts are needed to develop standardized formulas and compounding procedures for additional orphan active ingredients whose clinical efficacy is well-known but which are not available as products with a marketing authorization. Additionally, a legal framework at EU level is required to enable the full potential of pharmaceutical compounding for orphan active ingredients.

Effect of chlorination by-products on the quantitation of microcystins in finished drinking water.

Microcystins are toxic peptides that can be produced by cyanobacteria in harmful algal blooms (HABs). Various analytical techniques have been developed to quantify microcystins in drinking water, including liquid chromatography tandem mass spectrometry (LC/MS/MS), enzyme linked immunosorbent assay (ELISA), and oxidative cleavage to produce 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) with detection by LC/MS/MS, the "MMPB method". Both the ELISA and MMPB methods quantify microcystins by detecting a portion of the molecule common to most microcystins. However, there is little research evaluating the effect of microcystin chlorination by-products potentially produced during drinking water treatment on analytical results. To evaluate this potential, chlorinated drinking water samples were fortified with various microcystin congeners in bench-scale studies. The samples were allowed to react, followed by a comparison of microcystin concentrations measured using the three methods. The congener-specific LC/MS/MS method selectively quantified microcystins and was not affected by the presence of chlorination by-products. The ELISA results were similar to those obtained by LC/MS/MS for most microcystin congeners, but results deviated for a particular microcystin containing a variable amino acid susceptible to oxidation. The concentrations measured by the MMPB method were at least five-fold higher than the concentrations of microcystin measured by the other methods and demonstrate that detection of MMPB does not necessarily correlate to intact microcystin toxins in finished drinking water.

Transformation of microcystins to 2-methyl-3-methoxy-4-phenylbutyric acid by room temperature ozone oxidation for rapid quantification of total microcystins.

Microcystins (MCs) are cyanobacterial hepatotoxins capable of accumulation into animal tissues. To determine the total microcystins in water, a novel analytical method, including ozonolysis, methylation of 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) with methylchloroformate (MCF) and gas chromatography mass spectrometry (GC-MS) detection was developed. The results show that MCs can be oxidized by ozone to produce MMPB at ambient temperature, proving ozonation is an effective, rapid and green method for the transformation of MCs to MMPB without secondary pollution. The oxidation conditions as well as the esterification process were optimized and, subsequently applied to analysis of environmental samples. The method shows wide linear range and high sensitivity with a detection limit of 0.34 µg L(-1). The established method was successfully applied to the analysis of microcystins in water samples.

Using the MMPB technique to confirm microcystin concentrations in water measured by ELISA and HPLC (UV, MS, MS/MS).

Microcystins have been detected in raw and finished drinking water using a variety of techniques, including assays (immunoassay, phosphatase inhibition) and HPLC (UV, MS/(MS)). The principal challenge to microcystin analysis is accounting for the over 150 variants that have been described. A confirmatory individual variant HPLC analysis is prone to under-reporting total microcystins due to method specificity. One method that allows for total microcystin quantitation is the MMPB technique. In this study, water samples with native microcystins were oxidized to cleave the Adda moiety, common to all microcystin variants. LC-MS/MS analysis was conducted on the subsequent MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) molecule and calibrated using a certified reference standard (microcystin-LR) and 4-phenylbutyric acid. Total microcystin concentrations from MMPB were compared to Adda ELISA and individual variant analyses (LC-UV, LC-MS/(MS)). Variants of microcystin, including [DAsp(3)]MC-RR, [Dha(7)]MC-RR, MC-RR, MC-YR, MC-LR, [DAsp(3)]MC-LR, [Dha(7)]MC-LR, MC-WR, MC-LA, and MC-LY were detected and quantified in samples. The individual variant analyses did not account for total microcystins present in samples, as indicated by ELISA and MMPB data. Results demonstrated the MMPB technique is a simple and valuable approach to confirm ELISA data when analyzing microcystins, with method detection limits of 0.05 µg L(-1) for total microcystins.

Detection, quantification, and total synthesis of novel 3-hydroxykynurenine glucoside-derived metabolites present in human lenses.

PURPOSE: 3-Hydroxykynurenine O-ß-D-glucoside (3OHKG) protects the lens from UV damage, and novel related species may act analogously. The aim of this study was to detect, quantify, and elucidate the structures of novel 3-hydroxykynurenine glucoside-derived metabolites present in the human lens. METHODS: Compounds were detected and quantified by liquid chromatography with tandem mass spectrometry (LC-MS/MS) in 24 human lenses of different ages, of which 22 were normal and two had cataract. Structures of these were confirmed through total synthesis. RESULTS: 3OHKG concentrations decreased with age in the lens nuclei, whereas the levels of three novel species, 4-(2-amino-3-hydroxyphenyl)-2-hydroxy-4-oxobutanoic acid O-ß-D-glucoside (3OHKG-W), 3-hydroxykynurenine O-ß-D-glucoside yellow (3OHKG-Y), and 2-amino-3-hydroxyacetophenone O-ß-D-glucoside (AHAG), increased, though to different extents. In contrast, the concentrations present in the cortex of the lens remained constant with age. CONCLUSIONS: Three novel 3OHKG-derived metabolites have been detected in extracts from human lenses.

Determination of phenylbutyric acid and its metabolite phenylacetic acid in different tissues of mouse by liquid chromatography with tandem mass spectrometry and its application in drug tissue distribution.

Endoplasmic reticulum (ER) stress is associated with various human diseases. Phenylbutyric acid (PBA) is a well-known chemical chaperone that regulates ER stress. The main objective of this study was to develop a simple, rapid, and sensitive method for the simultaneous determination of phenylbutyric acid and its metabolite, phenylacetic acid (PAA). A LC-MS/MS analysis using negative electrospray ionization was used. Samples were analyzed by multiple reaction monitoring (MRM) in 15 min of total run time, using d11-PBA and d7-PAA as internal standards. The limit of quantification was 1 µg/g for tissue and 0.8 µg/mL for plasma. Recoveries for plasma and tissues were higher than 81% for both PBA and PAA. The inter-day and intra-day accuracy and precision were within ±15%. We then further successfully validated this method by applying it to determine the tissue distribution of PBA and its metabolite PAA after i.p. injection of PBA at a dose of 500 mg/kg in mice. The maximum concentrations of PBA and PAA in plasma and tissues were seen at 15 min and 45 min, respectively. The PBA plasma concentration was 15-fold higher than the concentration in the kidney, whereas the PAA plasma concentration was 6-fold higher than the concentration in the liver. The area under the curve decreased in the order of plasma > kidney > liver > heart > muscle > lung for PBA and plasma > liver > kidney > heart > muscle > lung for PAA. The tissue to plasma ratio ranged from 0.007 to 0.063 for PBA and 0.016 to 0.109 for PAA. In summary, the LC-ESI-MS method developed in this study is simple, sensitive and reliable.

Optimal strategies for determination of free/extractable and total microcystins in lake sediment.

The optimization of analytical procedures for the quantification of free and total microcystins (MCs) in natural sediments was systematically examined based on solvent extraction and Lemieux oxidation. In this optimized analytical procedure, a sequential solvent extraction using 50% (v/v) methanol and EDTA-sodium pyrophosphate was selected as the optimal extraction solvent for free MCs analysis, after which the purified extracts and sediment residuals were applied to the optimized Lemieux oxidation for determination of total MCs in lake sediments. The optimized procedures were shown to be efficient and reliable for the routine analysis of both free and total MCs in lake sediment samples, as indicated by the minimal adverse impact of sediment organic matter on the recovery of free MCs and yield of MMPB (2-methyl-3-methoxy-4-phenylbutyric acid). Finally, the developed procedures were applied to field sediment samples collected from Lake Dianchi during a bloom season and seven of thirty samples showed positive results.

Rapid quantification of total microcystins in cyanobacterial samples by periodate-permanganate oxidation and reversed-phase liquid chromatography.

Microcystins (MCs) comprise a family of more than 80 related cyclic hepatotoxic heptapeptides. Oxidation of MCs causes cleavage of the chemically unique C20 beta-amino acid (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) amino to form 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB), which has been exploited to enable analysis of the entire family. In the present study, the reaction conditions (e.g. concentration of the reactants, temperature and pH) used in the production of MMPB by oxidation of cyanobacterial samples with permanganate-periodate were optimized through a series of well-controlled batch experiments. The oxidation product (MMPB) was then directly analyzed by high-performance liquid chromatography with diode array detection. The results of this study provided insight into the influence of reaction conditions on the yield of MMPB. Specifically, the optimal conditions, including a high dose of permanganate (> or = 50 mM) in saturated periodate solution at ambient temperature under alkaline conditions (pH approximately 9) over 1-4 h were proposed, as indicated by a MMPB yield of greater than 85%. The technique developed here was applied to determine the total concentration of MCs in cyanobacterial bloom samples, and indicated that the MMPB technique was a highly sensitive and accurate method of quantifying total MCs. Additionally, these results will aid in development of a highly effective analytical method for detection of MMPB as an oxidation product for evaluation of total MCs in a wide range of environmental sample matrices, including natural waters, soils (sediments) and animal tissues.

Ionic liquid-based dynamic liquid-phase microextraction: application to the determination of anti-inflammatory drugs in urine samples.

Dynamic liquid-phase microextraction (dLPME) using an ionic liquid as acceptor phase is proposed for the determination of six non-steroidal anti-inflammatory drugs (NSAIDs) in human urine samples for the first time. The extraction is carried out in a simple and automatic flow configuration. The chemical affinity between the extractant (1-butyl-3-methylimidazolium hexafluorophosphate) and the analytes permits a selective isolation of the drugs from the sample matrix allowing also their preconcentration. The whole analytical method has been optimized taking into account all the chemical, physical and hydrodynamic variables. The proposed method is a valuable alternative for the analysis of these drugs in urine within the concentration range 0.1-10 microg mL(-1), allowing their determination at therapeutic and toxic levels. Limits of detection were in the range from 38 ng mL(-1) (indomethacin) to 70 ng mL(-1) (naproxen). The repeatability of the proposed method expressed as RSD (n=5) varied between 2.1% (flurbiprofen) and 3.8% (tolmetin).

Metabolite identification using a nanoelectrospray LC-EC-array-MS integrated system.

A novel approach to the parallel coupling of normal-bore high-performance liquid chromatography (LC) with electrochemical-array detection (EC-array) and nanoelectrospray mass spectrometry (MS), based on the use of a nanosplitting interface, is described where both detectors are utilized at their optimal detection mode for parallel configuration. The dual detection platform was shown to maintain full chromatographic integrity with retention times and peak widths at half-height between the EC-array and MS displaying high reproducibility with relative standard deviations of <2%. Detection compatibility between the two detectors at the part per billion level injected on-column was demonstrated using selected metabolites representative of the diversity typically encountered in physiological systems. Metabolites were detected with equal efficiency whether neat or in serum, demonstrating the system's ability to handle biological samples with limited sample cleanup and reduced concern for biological matrix effects. Direct quantification of known analytes from the EC-array signal using Faraday's law can eliminate the need for isotopically labeled internal standards. The system was successfully applied to the detection and characterization of metabolites of phenylbutyrate from serum samples of Huntington's disease patients in an example that illustrates the complementarity of the dual detection nanoelectrospray LC-EC-array-MS system.

Applications of derivative UV spectrophotometry for the determinations of fenbufen and ketoprofen in pharmaceutical preparations.

A methods for the determination of fenbufen and ketoprofen in pharmaceuticals by classical spectrophotometry--zero order derivative, first and second order derivatives spectrophotometry is described. using "peak-peak" (P-P) and, "peak-zero" (P-O) measurements. The calibration curves are linear within the concentration range of 2.0 - 12.0 microg x mL(-1) for fenbufen and ketoprofen. The procedure is simple, rapid and the results are reliable.

High performance liquid chromatographic determination of oxeladin citrate and oxybutynin hydrochloride and their degradation products.

Two high performance liquid chromatographic (HPLC) methods are presented for the determination of oxeladin citrate (OL) and oxybutynin hydrochloride (OB) and their degradation products. The first method was based on HPLC separation of OL from its degradation product using a Nucleosil C(18) column with a mobile phase consisting of acetonitrile -0.1% phosphoric acid (60:40 v/v). The second method was based on HPLC separation of OB from its degradation product using a VP-ODS C(18) column with a mobile phase consisting of acetonitrile/0.01 M potassium dihydrogen phosphate/diethylamine (60:40:0.2). Quantitation was achieved with UV detection at 220 nm based on peak area. The two HPLC methods were applied for the determination of OL or OB, their degradation products, methylparaben and propylparaben in pharmaceutical preparations. The proposed methods were used to investigate the kinetics of acidic and alkaline degradation processes of OL and OB at different temperatures and the apparent pseudofirst-order rate constant, half-life and activation energy were calculated. The pH-rate profiles of degradation of OL and OB in Britton-Robinson buffer solutions within the pH range 2-12 were studied.

LC procedure with SPE for quantification of indobufen enantiomers: pharmacokinetic studies.

A rapid and selective liquid chromatography (LC) with solid phase extraction (SPE) to quantify indobufen (INDB) enantiomers is described. The INDB enantiomers and internal standard (racemic flurbiprofen) are extracted from a small volume of acidified serum (0.2 ml) by means of SPE using cartridges with octadecyl chemically bound phase and analysed on reversed phase (RP), C18 column with ultraviolet detection at 275 nm. Recovery of both INDB enantiomers was in the range 92.1-94.3%. The mobile phase is composed of acetonitrile-potassium dihydrogen phosphate (pH 4.75; 10 mM) (38:62, v/v). The linear range of the standards curves was from 0.25 to 25.00 microg ml(-1) in serum for both enantiomers. The limit of quantification and detection for both INDB enantiomers in serum were 0.25 microg ml(-1) (CV < or = 10%), and 0.1 microg ml(-1), respectively. Both intra- and inter-day accuracy and precision of the calibration curves were determined and their CV values did not exceed 10%. The validated method has been successfully applied for chiral pharmacokinetics studies of INDB from tablets and intramuscular injections in man.

In vitro and ex vivo anti-human immunodeficiency virus (HIV) activities of a new water-soluble HIV protease inhibitor, R-87366, containing (2S,3S)-3-amino-2-hydroxy-4-phenylbutanoic acid.

In a series of compounds containing (2S,3S)-3-amino-2-hydroxy-4-phenylbutanoic acid (AHPBA), a transitionstate mimetic, R-87366:(2S,3S)-3-[N-(quinoxaline-2-carbonyl)-L-asparaginyl]amino- 2-hydroxy-4-phenylbutanoyl-L-proline tert-butylamide, was found to be a potent human immunodeficiency virus protease inhibitor (Ki value was 11 nM) and anti-HIV agent (IC90 value was 0.5 microM for HIV-1IIIB acutely infected cells) with moderate water-solubility (4.2 mg/ml at 25 degrees C). The compound was also active in chronically infected Molt-4/HIV-1IIIB cells, and inhibited the proteolytic processing of p55 into p17, suggesting that its anti-HIV activity was derived from HIV protease inhibition. The compound showed more potent activity (IC90 value was 0.03-0.25 microM) against clinical isolates of HIV in 5 out of 6 patients examined with varying clinical status in an ex vivo assay. One isolate, however, from the sixth patient, was less sensitive to R-87366 (IC90 value was 0.5 microM). In experiments with this strain, R-87366 showed comparatively low efficacy in acutely infected peripheral blood mononuclear cell (PBMC). This result suggests that the diversity of sensitivity shown in the ex vivo assay could be caused by the viral property itself. As a result of the determination of nucleic acid sequences in the clinical isolates, some amino acids were found to be substituted in the protease region, in contrast to the HIV-1 clade B consensus sequence, and some of them have been reported to contribute to the susceptibility of HIV protease inhibitors.

Mass spectrometric screening method for microcystins in cyanobacteria.

A screening method for microcystins in cyanobacteria, which consists of the formation of 3-methoxy-2-methyl-4-phenylbutyric acid as an oxidation product of microcystins by ozonolysis, and detection of 3-methoxy-2-methyl-4-phenylbutyric acid by thermospray-liquid chromatography/mass spectrometry or electron ionization-gas chromatography/mass spectrometry using selected ion monitoring, was developed. The ozonolysis made it possible to significantly reduce the formation times of 3-methoxy-2-methyl-4-phenylbutyric acid because the previously required extraction, clean-up and other procedures could be entirely eliminated. The resulting intact 2-methyl-4-phenylbutyric acid was directly analyzed by thermospray-liquid or electron ionization-gas chromatography/mass spectrometry, and the procedures from ozonolysis to analysis of microcystins at the pmole levels were performed within only 30 min. The calibration curves obtained by thermospray-liquid or electron ionization-gas chromatography/mass spectrometry analysis showed a linear relationship from 14 to 830 pmole and from 2.5 to 100 pmole of microcystin-LR, respectively. The method was applied to the detection and determination of the total amount of microcystins in bloom and cultured samples, showing that it provided a means of not only screening for microcystins but of their accurate quantitation.

Design and synthesis of substrate-based peptidomimetic human immunodeficiency virus protease inhibitors containing the hydroxymethylcarbonyl isostere.

The human immunodeficiency (HIV) codes for an aspartic protease known to be essential for retroviral maturation and replication. The HIV protease can recognize Phe-Pro and Tyr-Pro sequences as the virus-specific cleavage site. These features provided a basis for the rational design of selective HIV protease-targeted drugs for the treatment of acquired immunodeficiency syndrome (AIDS). HIV protease is formed from two identical 99 amino acid peptides. We replaced the two Cys residues by L-Ala to synthesize [Ala67,95]-HIV-1 protease by the solid phase method and then prepared [Tyr6,42, Nle36,46, (NHCH2COSCH2CO)51-52, Ala67,95] HIV-1 protease (NY-5 isolate) using the thioester chemical ligation method. Based on the substrate transition state, we designed and synthesized a novel class of HIV protease inhibitors containing an unnatural amino acid, (2S, 3S)-3-amino-2-hydroxy-4-phenylbutyric acid, named allophenylnorstatine (Apns) with a hydroxymethylcarbonyl (HMC) isostere. Among them, the conformationally constrained tripeptide kynostatin (KNI)-272 (iQoa-Mta-Apns-Thz-NHBut) was a highly selective and superpotent HIV protease inhibitor (Ki = 0.0055 nM). KNI-272 exhibited potent antiviral activities against both AZT-sensitive and -insensitive clinical HIV-1 isolates as well as HIV-2 with low cytotoxicity. After i.d. administration, bioavailability of KNI-272 was 42.3% in rats. Also, KNI-272 exhibited in vivo anti-HIV activities in human PBMC-SCID mice. The x-ray crystallography and molecular modeling studies showed that the HMC group in KNI-272 interacted excellently with the aspartic acid carboxyl groups of HIV protease active site in the essentially same hydrogen-bonding mode as the transition state. This result implies that the HMC isostere is an ideal transition-state mimic and contributes to the high activity of KNI-272.

High pressure liquid chromatographic determination of the new non-steroidal anti-inflammatory agent butibufen.

High performance liquid chromatography (HPLC) method for the determination and validation of a new non-steroidal anti-inflammatory agent, butibufen (CAS 55837-18-8), microencapsulated butibufen and its pharmaceutical forms--tablets, sachets (microencapsulated butibufen with excipients), microemulsion and cream--is described. Acetonitrile proved to be the best solvent for the extraction of butibufen from its pharmaceutical forms. The results submitted in this paper suggest that the HPLC method for the quantitative determination of butibufen is linear, accurate, precise and sensitive.

High-performance liquid chromatographic method for direct resolution of the indobufen enantiomeric components.

A high-performance liquid chromatographic method for the determination of (S)-(+)- and (R)-(-)-indobufen as diastereoisomeric derivatives has been applied and validated for stability control in tablet formulations. In order to obtain a less troublesome and more rapid method the direct resolution of the enantiomeric components by chiral stationary phases was investigated. The direct separation of the enantiomers was achieved using the cellulose tris(3,5-dimethylphenylcarbamate) derivative Chiralcel OD as a chiral stationary phase. The method was validated for the determination of the optical purity of (S)-(+)-indobufen as a quality control method for finished dosage forms.