Changes in the ultraviolet absorption spectra with pH in aqueous solution were used to determine the pKA values of amidines. A new mathematical procedure was used to study sparingly soluble compounds. In this technique, all the experimental data are used to solve for the absorbance of the insoluble basic species and for the pKA value by a 'complex method' of optimization, which is more general than the classical least-squares method. A relationship between a double bond vibration frequency and the acidity constants of these compounds has been demonstrated.
The analysis of pharmacologically active compounds using electrochemical techniques with modified electrodes is reviewed. Several physical and chemical methods for the immobilization of materials on to electrodes are available, and the resulting surfaces can be examined microscopically . Examples of these procedures and their analytical applications are given.
Polymorphism in the evolution of quality control of drugs is discussed and the possible relationship between polymorphism and bioavailability of drugs demonstrated. The advantages and limitations of the techniques used for the characterization of polymorphs are described.
The main purpose of this presentation is to define the different situations in which dissolution tests are used. The criteria for choosing a method and the establishment of the experimental procedure depend on the research goal. Information concerning the dissolution medium (surfactants, enzymes, volume, de-aeration) confirms, disproves, or completes the classical recommendations.
Determination of drugs in foods. In foods, drugs can come from different sources: animal therapeutics, zootechnical feed supplementation or human food additives. The possible consequences arising from the presence of drugs in food are considered. The quantitative determination of drugs in feeds or in human foods must fulfil various requirements, including specificity, sensitivity, accuracy and rapidity. Spectroscopic, electrochemical and chromatographic techniques are discussed. Some examples reported are ipronidazole, pyrimethamine, diethylstilboestrol, decoquinate and chloramphenicol. The need for quality assurance in analysis is also discussed.
An isocratic reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for the determination of albuterol sulfate and six of its related substances in albuterol sulfate inhalation solution, 0.5% (w/v). The separation was achieved using a YMC phenyl column (250 mm x 4.6 mm ID, 5 microm fitted with a direct connect YMC phenyl guard column (20 mm x 4 mm ID) maintained at ambient conditions, and a mobile phase of 25 mM monobasic potassium phosphate (pH 3.0) and methanol (95:5, v/v). The mobile phase flow rate was 1.5 mL/min and the detection wavelength was 225 nm. Albuterol is quantitated versus an external standard. The method was capable of resolving six of the seven known albuterol-related substances. Bis-ether albuterol, a drug substance process related impurity, is retained on the column due to its different hydrophilic character. The related substances are determined by area percent. However, a correction factor of 1.6 is applied for the determination of albuterol aldehyde, a potential impurity and a degradation product, since its molar absorptivity is about 1.6 times that of albuterol. The limits of detection and quantitation for albuterol and six of its related substances ranged between 0.01 and 0.21% of the assay concentration of 0.3 mg/mL as albuterol base. The method was found to be linear for albuterol over the range of 50-150% of the active label claim. The method was also found to be linear for the six related substances over the range 0.05-0.5%. No interferences from the blank, placebo (formulation matrix), related substances or force-degraded placebo samples were observed for the determination of the active or the individual related substances. The method was found to be accurate, precise, linear, specific, sensitive, rugged, robust, and stability-indicating.
D-002 is a new natural product consisting of a mixture of aliphatic fatty alcohols, which shows antioxidant and anti-ulcer effects in experimental models. A new validated methodology for determining simultaneously residual n-hexane and acetone in D-002 using the headspace gas chromatography (HS/GC) is described. The very poor solubility of D-002 in most solvents did necessary sample preparations in solid state. Limit test conditions allowed a detection of residual n-hexane and acetone more sensitively than that recommended for such purposes in the general method of the European Pharmacopoeia. Validation assays, applied to both D-002 residual solvents, proved: suitable sensitivity; very high linearity (correlation coefficients > or =0.999, R.S.D. of slopes < or =0.8% and R.S.D. of response factors < or =5% and no biases) and accuracy (average recoveries between 94.7 and 100.1%); and precision was < or =2.1%. The method was found suitable for quality control and stability studies of this new product.
Ganciclovir is an antiviral nucleoside analogue approved for treatment and prevention of cytomegalovirus infections in immunocompromised subjects. RS-79070-194, a diastereomeric monovalyl ester of ganciclovir (hydrochloride salt), is under evaluation as a prodrug to increase the bioavailability of ganciclovir. An HPLC method with column switching has been developed and validated for quantification of the corresponding free base RS-79070-004 in human plasma. In the method, proteinaceous material in 0.25 ml of plasma is precipitated by trichloroacetic acid. An aliquot of the supernatant is analyzed by HPLC, with automated column switching to remove late-eluting materials that might interfere with the analyte peaks in subsequent runs. Detection of RS-79070-004 is by UV lambda = 254 nm). The peak areas for each isomer are summed to generate a value for total RS-79070-004. The method has a validated range of 0.0400-4.00 microg/ml and a lower limit of quantification of 0.0400 microg/ml. All intra- and inter-assay %CVs were < 7.5%, and all recoveries (accuracy) were within 6% of nominal values. No interference was observed by ganciclovir, caffeine, acetaminophen, or ibuprofen. Analyte stability in plasma and in the sample extracts is adequate for the specified collection, storage, and analysis conditions. The validated method has been successfully used to analyze clinical study samples.
ON 01210.Na is a chlorobenzylsulfone derivative with potential property to mitigate the effects of accidental or intentional exposure to life threatening levels of radiation. A simple and sensitive HPLC method was developed and validated for the assay of ON 01210.Na. The isocratic system used a mobile phase consisting of acetonitrile:0.1% trifluroacetic acid in water (60:40, v/v) at a flow rate of 1 ml/min. The method used a C-18 Gemini column (250 mm x 4.6 mm) with column effluents monitored at 254 nm. Forced degradation of the drug was achieved by autoclaving ON 01210.Na with 0.05 N HCl, 0.05 N NaOH or 1.5% (v/v) hydrogen peroxide. The assay validation parameters evaluated include specificity, linearity, precision, accuracy and sensitivity. The retention time of the drug and the other effluents were well within 7 min. Standard curves were linear over the concentration range of 10-500 microg/ml. The R.S.D. values for the within-day and day-to-day precision ranged from 0.4 to 2.5 and 2.2 to 4.4%, respectively. The R.S.D. for accuracy measurement ranged from 0.85 to 1.7%. The critical level, the detection level and the determination level for this assay were 2.86+/-0.67, 5.69+/-0.67 and 15.6+/-1.8 microg/ml, respectively. A simple, sensitive and stability indicating HPLC assay was developed and validated for the analysis of a novel radioprotectant. This method was used to evaluate the aqueous as well as solid-state stability of this drug during autoclaving.
Axitinib (AG-013736) is a potent investigational drug that has antitumor activity in patients with metastatic renal cell carcinoma and other types of cancers. In this study, ion mobility spectrometry and "direct analysis in real time" (DART™) mass spectrometry were used to rapidly identify AG-013736 in drug substance samples and 1mg Axitinib tablets. The plasmagrams of the sample solutions exhibited a major peak with a reduced ion mobility that was within ±0.0002cm(2)V(-1)s(-1) of that for AG-013736 in an external reference standard solution. The DART ionization source was coupled with both a time-of-flight mass spectrometer and a lower-resolution ion trap mass spectrometer. Samples were analyzed by this technique in as little as 5s with minimal to no sample preparation required. The isotopic masses of the protonated dimer ions of AG-013736 were used to identify AG-013736 in the active tablet. Both techniques were also used to develop low-level limit tests for rapidly verifying the presence or absence of AG-013736 in blinded clinical supplies of active and matching placebo tablets of Axitinib.
The emodin-involved hepatotoxicity has been gaining increasing attention. The purpose of the present study was to evaluate the cytotoxicity of emodin on cultured human liver cells (L-02) and predict the possible relation between its cytotoxicity and cellular toxicokinetics. Cell viability and cell damage were assessed by Cell Counting Kit-8 (CCK-8) assay and phase-contrast microscopy, respectively. Cytotoxicity tests demonstrated a concentration- and time-dependent toxic effect of emodin on L-02 cells. Furthermore, emodin at concentration of 30μM led to a significant apoptosis in a time-dependent manner supported by the morphological changes of drug-treated cells. In addition, to elucidate the toxicokinetic characteristics of emodin, a highly sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method was employed and validated for detecting the dynamic alteration of emodin in cells and cell culture media. The proposed method appeared to be suitable for the analysis of emodin with desirable linearity (r(2)>0.99), and satisfying precision being less than 8.7%. The range of recoveries of this method was 90.2-101.9%. The preliminary cellular toxicokinetic study revealed a time-dependent intracellular accumulation of emodin, which was consistent with its in vitro toxic effects. These findings confirmed the cytotoxicity of emodin against L-02 cells and displayed the cytotoxic manner of emodin in terms of its cellular uptake and accumulation in L-02 cells.
N-[6-[2-[(5-bromo-2-pyrimidinyl)oxy]ethoxy]-5-(4-methylphenyl)-4-pyrimid inyl]-4-(2-hydroxy-1, 1-dimethylethyl) benzenesulfonamide sodium salt (TA-0201) is a novel orally active non-peptide antagonist for endothelin (ET) receptors. A sensitive and simultaneous determination method of TA-0201 and its major metabolites by liquid chromatography tandem mass spectrometry (LC--MS/MS) in a selected reaction monitoring mode using [2H6]TA-0201 as the internal standard was developed, and the plasma and tissue concentrations of TA-0201 and its major metabolites were determined in a pharmacokinetic study. The lower limit of determination of plasma TA-0201 concentrations by this method was 0.1 ng/0.5 ml, and the between- and within-run accuracy and precision were both less than 5.1%, in the calibration curve range of 0.1-50 ng/0.5 ml. This method was applied to determine the concentrations of TA-0201 and its metabolites in plasma and various target tissues, i.e. the heart, lung and kidney, after oral administration of TA-0201 (0.1 mg/kg(-1)) to male rats. TA-0201 and its major metabolite of a carboxylic acid form were detected in plasma and all the tissues 24 h after administration, their tissue concentrations being higher than those in plasma and still detectable at 72 h. Thus, this method could successfully be applied to study pharmacokinetic properties of TA-0201 in rats.
A highly sensitive LC method has been developed and validated for quantitation of Ro 24-0238 in human plasma using Ro 24-2446 as an internal standard. With 1 ml of plasma, the limit of quantitation of the method was 50 pg ml-1 of Ro 24-0238. After solid-phase extraction with C18 reversed-phase cartridges, the samples were reconstituted in an acidic buffer solution; under these conditions, Ro 24-0238 and Ro 24-2446 (IS) were converted to their cationic forms. The LC system employed a strong cation exchange column and a narrow bore reversed-phase column, connected via a column switching valve. The cationic analyte and internal standard were separated from most of the endogenous components of plasma on the cation exchange column. A small fraction containing the analyte and the internal standard was transferred by automated valve switching to the narrow bore reversed-phase column, which further resolved the individual components. The chromatography was monitored by UV absorption at 322 nm. The overall intra-assay precision was 3.6% (RSD) and the per cent error was less than +/- 11%. The overall inter-assay precision was 3.9% (RSD). Linearity was demonstrated in a concentration range of 50-5000 pg ml-1. This method has been applied to pharmacokinetic studies of Ro 24-0238 in man.
Phase II attrition of clinical candidates in the drug development cycle is currently a major issue facing the pharmaceutical industry. To decrease phase II attrition, there is an increased emphasis on validation of mechanism of action, development of efficacy models and measurement of drug levels at the site of action. PD 0332991, a highly specific inhibitor of cyclin-dependent kinase 4 (CDK-4) is currently in clinical development for the treatment of solid tumor. A clinical presurgical study will be required to better understand how PD 0332991 affects signaling pathways and how the intratumoral concentration of PD 0332991 correlates with plasma PK parameters and molecular alterations in breast cancer tissues after PD 0332991 treatment. Before conducting such a clinical study, it is important to evaluate PD 0332991 levels in tumor tissue samples from a xenograft mouse model for the determination of drug exposure at the site of action. Therefore, the objectives of this study were (1) to develop and validate a sensitive LC-MS/MS method to quantify PD 0332991 in mouse tumor tissues from MDA-MB-231-Luc human breast tumor xenografts in SCID-beige mice; (2) to quantify PD 0332991 levels in mouse tumor tissues after oral administration of PD 0332991 at 10 and 100mg/kg using the validated LC-MS/MS method. Both liquid-liquid extraction (LLE) and supported liquid extraction (SLE) in a 96-well format were developed and evaluated to achieve optimal extraction recovery with minimal matrix effects. The newly developed SLE method is more efficient (speed and ease) and demonstrates comparable recovery (93.1-100% at three different concentrations) compared to the traditional LLE method. The validated LC-MS/MS for PD 032291 in mouse tumor tissue homogenate method exhibited a linear dynamic range of 0.1-100 ng/mL with inter-day accuracy and precision within 15%. The validated method was successfully applied to measure PD 0332991 levels in tumor tissues in MDA-MB-231-Luc human breast tumor xenografts in SCID beige mice. The mean tumor concentrations at 6h post-oral PD 0332991 administration at 10 and 100mg/kg were 1793 (+/-1008) and 25,163 (+/-3959) ng/g, respectively.
Taranabant (MK-0364) is a highly potent and selective cannabinoid-1 receptor (CB-1R) inverse agonist. It is being developed at Merck & Company to treat obesity. The chemical synthesis of MK-0364 drug substance involved the direct coupling of chiral amine and pyridine acid side chains mediated by cyanuric chloride. Four major process impurities were observed and characterized using high performance liquid chromatography (HPLC) coupled with ultraviolet (UV) and electrospray ionization (ESI) mass spectrometry (MS) detectors. The exact mass data was used for structural elucidation which suggests that the impurities are derivatives of cyanuric chloride formed in the coupling step. Owing to the reactive nature of these impurities, an interesting degradation phenomenon was observed during stability testing of MK-0364 drug substance when stored at 40 degrees C/75% RH and 25 degrees C/60% RH conditions. Degradation pathways were proposed to explain the changes observed in the HPLC impurity profile. Forced degradation experiments were also conducted to confirm the degradation pathways and assess the stability of the impurities. Finally, the complete stability data of the bulk drug are reported to support the hypothesis.
A simple and accurate assay for quantitating MK-0476 [sodium 1-(((1(R)-(3-(2-(7-chloro-2-quinolinyl)-(E)-(ethenyl)phenyl)(3-(2-(1- hydroxy-1-methylethyl)phenyl)propyl)thio)-methyl)cyclopropane)acetate], which is a potent and selective leukotriene D4-receptor antagonist, in human plasma has been developed. The method involves precipitation of protein and reversed-phase liquid chromatography with fluorescence detection. The assay is linear in the range of 30-3000 ng ml-1 of MK-0476, and the limit of detection is 5 ng ml-1. The interday precision (% relative standard deviation) values of this method at 51 and 2040 ng ml-1 are 10 and 3%, respectively. The interday accuracy values at these concentrations are 94 and 104%, respectively. The absolute recovery of MK-0476 is 99%. The utility of this method to determine plasma concentrations of MK-0476 in humans receiving the drug orally was demonstrated.
A steoreoselective high-performance liquid chromatographic method was developed for the quantification of montelukast (free acid of Singulair, or MK-0476), a potent and selective leukotriene D4 (cysLT1) receptor antagonist, and it S-enantiomers (L-768,232). The method involves protein precipitation and fluorescence detection. Chromatographic separation of the enantiomers from endogenous components in plasma and chiral resolution of the enantiomers are achieved by using column switching HPLC and an alpha-acid glycoprotein chiral column. The assay is linear in the range of 28.9-386 ng ml-1 of free acids of montelukast and L-768,232. The intraday precision (% relative standard deviation) values of this method were in the range of 2.5-9.1% for montelukast, and 2.4-6.8% for L-768,232, while the intraday accuracy values were in the range of 97-103% for montelukast and 96-104% for L-768,232. The interday precision values of this method at 48.2 and 193 ng ml-1 were 5.3 and 3.6%, respectively, for montelukast, and 4.2 and 3.7%, respectively, for L-768,232, while the interday accuracy values at these concentrations were 97 and 103%, respectively, for montelukast and 99 and 102%, respectively, for L-768,232. The utility of the methodology was demonstrated by analysis of plasma samples from a study in which healthy volunteers received 10 mg per day of montelukast orally for 7 days. Results of this study indicate that there is no apparent bioinversion of montelukast to its S-enantiomer in humans.
An analytical method based on radioimmunoassay (RIA) has been developed for the determination of the antiarrhythmic agent, MK-0499, in plasma and urine. Owing to the potency of the drug, the specificity of this assay in human plasma could not be adequately determined using conventional RIA procedures. A highly specific procedure, based on LC/MS-MS, was developed to cross-validate the RIA. The lower quantifiable limits of the RIA and LC/MS-MS-based methods were 0.05 and 0.013 ng ml-1, respectively. Cross-validation data, compared using paired student's t-test regression analysis, showed excellent correlation between methods. The mass spectrometric assay was also used to simultaneously measure plasma concentrations of unlabeled and 14C-labeled MK-0499 following administration of the drug at high specific activity to volunteers.
A purified extract isolated from the dried flower buds of Magnolia fargesii (NDC-052) is currently being evaluated for phase III clinical trials as a new anti-asthma drug. A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the simultaneous determination of magnolin and epimagnolin A, the major bioactive components of NDC-052, in rat plasma was developed. After liquid-liquid extraction with tolterodine as an internal standard, magnolin and epimagnolin A were separated on a Luna phenyl-hexyl column with the mobile phase of 70% methanol in 10mM ammonium formate. The analytes were detected by an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curves were linear over the concentration range of 50-2500ng/mL for magnolin and epimagnolin A in rat plasma. The intra- and inter-day coefficients of variation and relative errors for magnolin and epimagnolin A at four QC concentrations were 1.5-11.4% and 5.9-12.5%, respectively. The lower limits of quantification for magnolin and epimagnolin A were 50.0ng/mL using 50microL of plasma. This method was successfully applied to the pharmacokinetic study of magnolin and epimagnolin A after an oral administration of NDC-052 in male Sprague-Dawley rats.
A simple high-performance liquid chromatographic method was developed for determination of a novel antineoplastic agent MKT-077 in plasma. MKT-077 was extracted from 50 microl of plasma with acetonitrile containing 1 ml trifluoroacetic acid per liter. Chromatographic separation was achieved within 13.5 min using a reverse-phase Puresil C18 analytical column. A visible detector operated at 490 nm was used. The linearity of the calibration curve was obtained (r2 = 0.99986) over the analytical range of 10-500 ng/ml(-1). The intra- and inter-assay precision was in the range of 0.9-11.1 and 0.3-4.4%, respectively. The intra- and inter-assay bias ranged from -7.3 to 11.1% and from 0.4 to 11.6%, respectively. The utility of this assay was demonstrated after the administration of a single dose of MKT-077 to rats. The plasma elimination half-life of MKT-077 was 1.8-4 h.
LC-NMR was applied to identify the polar volatile metabolite of MK-0869. MK-0869, a morpholine-based compound containing a triazolone ring, is a very potent NK(1) receptor antagonist. Currently, it is in development as an anti-emesis agent in chemotherapy treatments. The primary metabolites of MK-0869, M1 and M2, are non-polar and lack the triazolone ring. Incubation of [14C]M1 with liver microsomes from male rats produced a very polar and volatile metabolite, M3. Analysis was not possible by LC-MS or by conventional NMR because of poor ionization, small molecular weight and volatility, leaving chemical derivatization and LC-NMR as alternative methods. Reduction of M3 with NaBH(4) resulted in a derivative that had the same retention time as p-fluorophenylethylene glycol on HPLC. A small aliquot of the solution containing M3 was passed through the LC of the LC-NMR system, which was connected on-line with a radioactivity detector. The simultaneous UV and radioactivity chromatograms thus identified the chromatographic UV peak that was associated with the metabolite. Analysis was carried out by stop-flow on another portion of this fraction. From the chemical derivatization and the analysis by LC-NMR, M3 is shown to be p-fluoro-alpha-hydroxyacetophenone. Further studies using LC-NMR showed that M3 could be generated from both M1 and M2 in NADPH-dependant reactions catalyzed by microsomes containing recombinant human CYP2C19, CYP1A2 or CYP3A4.
A liquid-chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of GDC-0879 and its ketone metabolite (M1) in dog plasma to support preclinical toxicokinetic evaluation. The method consisted of solid phase extraction for sample preparation and LC-MS/MS analysis in positive ion mode using electrospray ionization for analysis. D(4)-GDC-0879 and (13)C(2)-D(2)-M1 were used as internal standards. A quadratic regression (weighted 1/concentration(2)) was used to fit calibration curves over the concentration range of 1-1000ng/ml for both GDC-0879 and M1. The accuracy (%bias) at the lower limit of quantitation (LLOQ) was 12.0% and 2.0% for GDC-0879 and M1, respectively. The precision (%CV) for samples at the LLOQ was 11.3% and 2.6% for GDC-0879 and M1, respectively. For quality control samples at 3.00, 400 and 800ng/ml, the between run %CV was ≤3.9% for GDC-0879 and ≤2.4% for M1. Between run %bias ranged from 4.6 to 12.0% for GDC-0879 and from -0.8 to 2.7% for M1. GDC-0879 and M1 were stable in dog plasma for at least 44 days at -70°C.
ABT-089 is a potent, selective neuronal cholinergic channel modulator with cognition enhancing activity in several animal paradigms. A simple and sensitive chromatographic method for the specific determination of ABT-089 in human plasma has been developed and validated. The method utilizes in situ precolumn fluorescence derivatization of the sample with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) prior to liquid-liquid extraction followed by reverse phase HPLC and fluorescence detection (lambda(ex) 495 nm, lambda(em) 533 nm). The described method significantly simplifies sample preparation. The derivatized product was separated from interference using a YMC ODS-AQ, 5 microm, 250x4.6 mm i.d. column using a mobile phase consisting of 30:5:65 (v:v:v) acetonitrile/methanol/aqueous buffer at a flow rate of 0.75 ml min(-1). The aqueous buffer consisted of 0.01 M tetramethylammonium perchlorate, 0.1% (v:v) trifluoroacetic acid, pH 3.0. An Alltech Absorbosphere CN, 5 microm, cartridge guard column was also used before the analytical column. Plasma samples were alkalinized with 0.1 M NaHCO3, 300 microl of a 1 mg ml(-1) ethanolic solution of NBD-F was added and the samples were heated in a water bath for 10 min at 50 degrees C. The samples were then extracted with tert-butylmethylether, evaporated to dryness and then reconstituted in mobile phase. For 1 ml of plasma, a limit of quantitation (LOQ) of 1.6 ng ml(-1) was obtained. The method was linear from 1.6 to 836 ng ml(-1). Inter and intra-day assay RSD (n = 6) were less than 9%. Accuracy determinations showed the quality control samples to range between 88-114% of the theoretical concentration.
TA-0910 [1-methyl-(S)-4,5-dihydroorotyl-L-histidyl-L-prolineamide] is a metabolically stable analogue of thyrotrophin-releasing hormone (TRH) and is under clinical investigation as a central nervous system function modulator. A method for determination of its plasma concentrations by radioimmunoassay (RIA) was established. TA-0910 was conjugated to bovine serum albumin and keyhole limpet haemocyanin (KLH) with bis-diazotized benzidine and 1,5-difluoro-2,4-dinitrobenzene as bridging agents. Anti-TA-0910 antisera were prepared by immunizing rabbits with the TA-0910 conjugates and Freund's complete adjuvant. The radiolabelled TA-0910 for RIA was prepared by introducing 125I into the histidine imidazole ring of TA-0910 by the Na125I/chloramine-T method, and purified by reversed phase high-performance liquid chromatography to give a specific radioactivity of 81.4 TBq mmole-1. As the result of testing the cross-reactivity of the antisera with assumed TA-0910 metabolites and TRH, a TA-0910-selective antiserum was obtained from a rabbit immunized with TA-0910-dinitrophenyl-KLH. RIA using this antiserum and the radiolabelled TA-0910 afforded a determination range of 10 pg approximately 5 ng ml-1 plasma. By using this RIA, the time courses of plasma concentrations of unchanged TA-0910 after oral and intravenous administration of TA-0910 were obtained in rats.
Radioimmunoassay (RIA) was investigated for the determination of TA-0910 and its main metabolite, TA-0910 acid-type, in human plasma and urine. TA-0910 is a new metabolically stable analogue of thyrotropin releasing hormone (TRH). Antiserum was raised in the rabbit against the 1-fluoro-2,4-dinitrophenyl derivative of TA-0910 or TA-0910 acid-type conjugated to keyhole limpet hemocyanin (KLH). The radioligand was prepared by iodination with 125I of the histidine imidazole ring of TA-0910 or TA-0910 acid-type. Cross-reactivities of anti-TA-0910 or TA-0910 acid-type antiserum for TA-0910, its metabolite and related compounds were low. The calibration range was 0.02-5 ng ml-1 using 100 microliters human plasma or urine. Inter-day variations of TA-0910 and TA-0910 acid-type assay in plasma were 3.5-15.5 and 1.8-9.4%, respectively. The variations of the assay in urine were the same as those in plasma. The recovery of TA-0910 and TA-0910 acid-type spiked in plasma or urine samples was approximately 100%. Furthermore, this method was applied to the determination of TA-0910 and TA-0910 acid-type in human plasma and urine samples, for the evaluation of the pharmacokinetics of TA-0910 in humans. From the results it was demonstrated that he developed RIA was useful for the determination of TA-0910 and TA-0910 acid-type in human plasma and urine, and was applicable to pharmacokinetic studies in humans.
A solid phase extraction (SPE) liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method for the determination of GDC-0941 concentrations in human plasma has been developed and validated to support clinical development. An Oasis MCX 10mg 96-well SPE plate was used to extract plasma samples (50 μL) and the resulting extracts were analyzed using reverse-phase chromatography and mass spectrometer coupled with a turbo-ionspray interface. The method was validated over the calibration curve range 0.500-500 ng/mL with linear regression and 1/x(2) weighting. Within-run relative standard deviation (%RSD) ranged from 1.5 to 11.5%, while the between-run %RSD varied from 0.0 to 4.4%. The accuracy ranged from 96.0% to 110.0% of nominal for within-run and 98.0% to 108.0% of nominal for between-run at all concentrations including the LLOQ quality control at 0.500 ng/mL. Extraction recovery of GDC-0941 was between 79.0% and 86.2%. Stability of GDC-0941 was established in human plasma for 602 days at -70 °C and 598 days at -20°C, respectively, and established in reconstituted sample extracts for 167 h when stored at room temperature. Internal standard normalized matrix factor was 1.1, demonstrating that the use of the stable-labeled internal standard GDC-0941-d(8) effectively compensated observed matrix effect and resulting in no adverse impact on the quality of the data produced. This assay was used for the determination of GDC-0941 human plasma concentrations over a sufficient time period to determine pharmacokinetic parameters at relevant clinical doses.
Two HPLC methods were developed: one for the quantitation of HBY 097 reverse transcriptase inhibitor and its metabolites M2 and M3 in human serum, and one for the quantitation of metabolite M5 in urine. The HPLC procedure for the quantitation of HBY 097 and its metabolites M2 and M3 in human serum involved protein precipitation with acetonitrile followed by automated on-line trace enrichment. The HPLC procedure for the analysis of metabolite M5 in urine involved enzymatic hydrolysis of urine with beta-glucuronidase to convert metabolite M5 (glucuronide of M3) to M3. Reverse phase chromatographic separation with gradient elution. UV detection at 335 nm, and internal standard were used to quantitate analytes in both procedures. The lower quantitation limits were 25 ng ml-1 for HBY 097 and metabolites M2 and M3 in serum, and 0.5 microgram ml-1 for the metabolite M5 in urine measured as metabolite M3 after hydrolysis. The HBY 097 and metabolite M3 concentrations were specific but metabolite M2 was semi-specific because the two diastereomers of M2 were not resolved by the present chromatographic procedure. Both procedures were applied to the quantitation of HBY 097 and its metabolites in serum and urine of HIV positive patients who were enrolled in a clinical study of drug safety and pharmacokinetics.
A quantitative method based on radioimmunoassay for the determination of the antifungal agent, CANCIDAS (MK-0991) has been developed and validated. The immunogen was prepared by coupling MK-0991 to bovine serum albumin through a two-step reaction with difluorodinitrobenzene. An antiserum specific to MK-0991 was selected for RIA. The assay was based on the competitive immunoassay principle in which the drug competes with iodinated drug for a limited quantity of specific antibody. The bound tracer was separated via goat anti-rabbit globulin. The assay demonstrates good accuracy and reproducibility at plasma concentration down to 10 ng/ml. The specificity of the RIA method was confirmed by cross-validating against an established HPLC method.
Analytical high-performance liquid chromatography (HPLC) methods using derivatized cellulose chiral stationary phases (CSPs) were developed for the separation of the enantiomers of 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide ((+/-) IDRA21). In previous studies, (+/-) IDRA21 has been found to have an interesting inhibitory effect on the desensitization of alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) receptor and improve cognition in animals. This compound possess one chiral carbon atom, but very little information has been reported on the stereoselectivity of his activity. Therefore resolution of the enantiomers of this compound and subsequent identification of stereospecificity in his pharmacological actions are clearly matters of interest. The resolution were made under normal- and reversed-phase conditions using a mobile phase consisting of n-hexane:2-propanol (70/30, v/v) and water:acetonitrile (60/40, v/v) respectively, and a CSP of silica-based cellulose tris-3,5-dimethyl-phenylcarbamate (Chiralcel OD and Chiracel OD-R). The enantiomeric nature of eluates was confirmed by circular dichroism (CD) spectra. A baseline separation (R(S) > 1.5) was obtained in both cases. Furthermore the isolation of optical isomers of (+/-) IDRA21 was performed using a semipreparative column packed with the same cellulose OD CSP.
The prototropic exchange equilibria of two drugs, nizatidine (I) and ranitidine(II), and also of structurally related the N,N'-dimethyl-2-nitro-1,1-ethenediamine molecule (III) were investigated. From the changes in electronic spectra in media of various acidity several protonation constants were determined. For pK values were -0.82, 1.95, and 6.67; for ranitidine pK values were 1.95 and 8.13; and for III was 2.60. The hydroxylation equilibrium constant in strongly alkaline media was determined too. Corresponding pK(a) values were 13.23 for I, 13.36 for II and 13.76 for III. Molecular orbital calculations of electronic spectra confirmed that pK 1.95 for I and II, and pK 2.60 for III, are associated with C-protonation of nitroethenediamine fragment, while all pK(a) values correspond to the addition of HO- anion at the same double bond.
A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay is reported for the determination of 4-amino-1-hydroxybutane-1, 1-diphosphonic acid (AHBuDP) monosodium salt trihydrate, a new inhibitor of bone resorption. The compound does not demonstrate any intrinsic UV properties and thus pre-column derivatization of the primary amino group of the drug with 9-fluorenylmethyl chloroformate (FMOC) at pH 9 in the presence of sodium citrate is required to facilitate UV detection of the analyte. Excess derivatization reagent is extracted with methylene chloride and an aliquot of the aqueous portion is assayed on a polymeric phase (Hamilton PRP-1) at 35 degrees C by reversed-phase HPLC. A mobile phase of 0.05 M citrate and 0.05 M phosphate buffer (pH 8.0)-acetonitrile-methanol (75:20:5, v/v/v) is utilized with UV detection at 266 nm. Application of the method to the analysis of AHBuDP in I.V. solution, tablet and capsule formulations is presented.
In this work, oxidative metabolism of the new propellant, 1,1,1,2-tetrafluoroethane to trifluoroacetic acid in man is shown to be minimal. Alternative propellants and refrigerants are under development to replace the currently used chlorofluorocarbons which lead to stratospheric ozone depletion. One potentially useful replacement is the hydrofluorocarbon, 1,1,1,2-tetrafluoroethane (HFA-134a). Before it can be used, however, particularly as a propellant in an aerosol pharmaceutical formulation whereby the compound is in effect dosed to people, it is important that the safety of this compound is established. As a part of this safety evaluation it is necessary to understand the metabolism of HFA-134a. In this work the production of the potential oxidative metabolite of HFA-134a, trifluoroacetic acid (TFA) has been studied in human urine following inhalation dosing with HFA-134a. The concentrations of TFA in urine have been measured using a highly sensitive 19F nuclear magnetic resonance procedure with a limit of detection of 10 ng ml-1 based on an acquisition time of only 2.25 h per sample. TFA is the only fluorinated species observed in the urine samples and only at very low levels, indicating that the oxidative route of metabolism can occur in vivo in man, but this metabolism is minimal in terms of percentage of administered dose.
A reversed-phase HPLC analytical method for the assay of 1,10-phenanthroline-5,6-dione (I) has been developed and validated. A C18 column (150 x 4.6 mm; 5 microm) was employed together with a mobile phase of methanol-water (50:50, v/v) containing 0.1% triethylamine. UV detection was performed at 254 nm. Dione (I) eluted as a spectrally pure peak resolved from its impurities allowing the method to be applied to the purity evaluation of samples obtained via two synthetic routes. In addition, 4,5-diazafluoren-9-one (V) was identified as the main impurity by employing the method in HPLC-MS mode with photodiode array UV detection.
A high performance liquid chromatographic (HPLC) procedure has been developed for the determination of cisplatin, based on the pre-column derivation of platinum(II) with reagent N,N'-bis(salicylidene)-1,2-propanediamine (H2SA2pn). The neutral platinum complex was extracted, concentrated in an organic solvent and then injected (5 microl) on a reverse phase HPLC column, Varian Micro-Pak SP C-18, 5 microm (150 mm x 4.0 mm i.d.). The complex was eluted isocratically using a ternary mixture of methanol/acetonitrile/water (40/30/30, v/v/v) at a flow rate of 1.0 ml/min and was determined by a UV detector set at 254 nm after elution. A detection limit was found to be 4.0 ng per injection. The amounts of platinum in blood serum and urine of cancer patients after administration of cisplatin were observed in a range of 221-298 ng/ml and 43-97 ng/ml with relative standard deviation (R.S.D.) of 3.6-4.6% and 3.5-4.8%, respectively. Preliminary metabolism profiles of Pt concentrations in blood and urine from the patients were established.
A HPLC method for determination of propane-1,2,3-triyl trinitrate and impurities: (2RS)-3-hydroxypropane-1,2-diyl dinitrate and 2-hydroxypropane-1,3-diyl dinitrate ointment was developed. The conditions for good separation of constituents, while avoiding vehiculum interference were established. The results feature of high accuracy and good precision. For individual constituents R.S.D. is ranged from 0.7 to 9.9%, while recovery was 100.1% for propane-1,2,3-triyl trinitrate and 95.1-99.0% for impurities. It has been found that propane-1,2,3-triyl trinitrate used in medicine in the form of ointment contains such impurities which can be identified and quantified at relatively low concentrations of 70 ng ml(-1).
A high performance liquid chromatography/electrospray ionization tandem mass spectrometric (HPLC/ESI MS/MS) method has been developed for quantification of pyrimido[1,2-a]purin-10(3H)-one adducts from DNA. The method is based on acid-catalyzed cleavage of the adducts from DNA and the use of [2,3a,10-13C3]pyrimido[1,2-a]purin-10(3H)-one as an internal standard in the analysis. For this purpose the latter compound was prepared. Rate constants for the acid-catalyzed cleavage of pyrimido[1,2-a]purin-10(3H)-one from the corresponding 2'-deoxyribonucleoside were determined, and its hydrolytic stability and possible formation by a cross reaction between guanine and [2,3a,10]pyrimido[1,2-a]purin-10(3H)-one were studied.
A new procedure is described for the selective determination of drugs containing a 1,2-diphenolic moiety. The assay is based upon the measurement of difference absorbance between two equimolar solutions of the drug in pH 7 phosphate buffer, one of which also contains 0.1 M boric acid. The difference absorbance, which is maximum at about 292 nm, is due to the different spectral characteristics of the boric acid ester of the drug and of the unesterified drug and is proportional to the concentration of the drug. The accuracy, precision, sensitivity and specificity of the procedure are discussed. Applications of the assay are described for adrenaline, isoetharine, isoprenaline, levodopa and methyldopa in pharmaceutical formulations.
The binary phase diagram of (S)-(+)-4,4'-(1-methyl-1,2-ethandiyl)-bis-(2,6-piperazinedione), 1 (dexrazoxane), a cardioprotective agent, and of its (R)-(-)-enantiomer, 2, has been investigated by differential scanning calorimetry (DSC); the equimolecular mixture of 1 and 2 corresponds to a racemic compound (racemate) whose melting point is higher than that of the enantiomers. Thermal behaviour (DSC) is examined and discussed in comparison with the data obtained by other physical methods (IR spectroscopy and X-ray powder diffraction).
A simultaneous assay of levodopa and benserazide in combined formulations is described that utilizes the different spectral properties of the drugs in the presence of germanium dioxide. Difference absorbances are measured at 238 and 292.5 nm between pH 6 solutions of the drugs complexed with germanium dioxide relative to equimolar solutions of the uncomplexed drugs. The concentrations are calculated using two simultaneous equations derived from the difference absorptivities of the individual drugs at 238 and 292.5 nm. The choice of wavelengths for maximum accuracy and precision is discussed. Good recoveries of the drugs in standard mixtures were obtained and satisfactory precision was demonstrated by the relative standard deviations of 0.46% and 1.07% for levodopa and benserazide, respectively, obtained in the replicate analyses of a sample of Madopar capsules. The accuracy of the procedure and the absence of interference from excipients were confirmed by the good agreement in the results for the assay of several batches of Madopar capsules and one of Madopar tablets with those obtained by the manufacturer by an HPLC procedure.
Sensitive and selective high performance liquid chromatographic (HPLC) methods for the quantification of 1,2-diethyl-3-hydroxypyridin-4-one (CP94), its iron complex [Fe(III) (CP94)3] and glucuronide metabolite (CP94-GLUC) in urine and serum of thalassaemic patients are described. Three separate analyses are involved. The first assay quantifies both CP94 and its iron complex. This procedure requires the conversion of the iron complex to the free ligand and is carried out using diethylenetriaminepentaacetic acid (DTPA). CP94 and the internal standard, 1-propyl-2-ethyl-3-hydroxypyridin-4-one (CP95) present in either serum or urine are then extracted at pH 7.0 with dichloromethane. Extraction efficiency is 96.0 +/- 5.6% and 100 +/- 7.1% for CP94 and CP95, respectively, and 31.2 +/- 2.1% at 30 microM and 53.2 +/- 4.2% at 300 microM for the corresponding iron complex. In the second assay, samples are incubated (16 h) with beta-glucuronidase and processed as before. In this assay, the drug, its iron complex and glucuronide conjugate are measured. In the third assay the iron complex of CP94, [Fe(III) (CP94)3] is quantified. From the three separate analyses it is possible to calculate the individual concentrations of the three separate components present in serum and urine of thalassaemic patients. Calibration for both components, i.e. CP94 (assays 1 and 2) and its iron complex (assay 3) are linear with correlation coefficients > 0.99 and are reproducible over the required concentration range of 0-500 microM for the free ligand and 0-100 microM for the iron complex. The minimum quantifiable level is 0.5 microM for the free ligand and 1.0 microM for the iron complex.
A sensitive, simple and rapid HPLC-MS/MS method has been developed and validated for the simultaneous determination of L-dopa and its prodrug (S)-4-(2-acetamido-3-ethoxy-3-oxopropyl)-1,2-phenylene diacetate (AEPD) in rat plasma in the present study. The analytes were separated on a C(18) column (5 microm, 2.1 mm x 150 mm) with a security guard C(18) column (5 microm, 4 mm x 20 mm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was applied for detection. With alpha-methyldopa as internal standard, sample pretreatment involved in a one-step protein precipitation with 0.4M perchloric acid. The method was linear over the concentration ranges of 50-5000 ng/ml for L-dopa and 12.5-2500 ng/ml for AEPD. The intra-day and inter-day relative standard deviations (RSD) were less than 15% and the relative errors (RE) were all within 15%. Finally, the method was successfully applied to support the pharmacokinetic study after L-dopa and its prodrug AEPD were orally administrated to the Sprague-Dawley rats, respectively.
A gas-liquid chromatographic method for the determination of feprazone in various biological matrixes, employing a choice of detector options, is described. After rapid, micro-scale extraction of the sample with n-butyl acetate at physiological pH, the solution was injected directly onto the chromatograph. Separation was with either an OV7 column and flame ionisation or electron capture detection, or with a carbowax high polymer column and nitrogen specific detection. When 100 microl of plasma was extracted the limit of accurate measurement was 2 mg 1(-1) for F.I.D. and N.P.D. and 0.5 mg 1(-1) with E.C. detection. Quantification was by comparison with a range of plasma calibrators carried throughout the procedure, and determination of peak height ratios against an internal standard incorporated into the extracting solvent. The CV of the assay throughout the concentration range normally encountered in patients undergoing feprazone treatment, ranged between 2.4 and 7.8% for the various detector options. The analytical method has been applied to samples collected both from patients and normal volunteers undergoing treatment with a range of feprazone maintenance doses.
An assay of berberine (BE) was developed with good selectivity and sensitivity based on the total internal reflected resonance light scattering (TIR-RLS) signals from water/1,2-dichloroethane (H(2)O/DCE) interface. Under optimal conditions, amphiphilic complex formed by BE and fluorescein (Flu) was adsorbed to H(2)O/DCE interface, resulting in good separation of BE from the coexisting foreign substances in aqueous phase and significant enrichment of BE at the interface. This enriched species at the interface was found corresponding to enhanced TIR-RLS signals located at 370.0 nm. Proportional relationships were established between the enhanced TIR-RLS intensity and the BE in the range of 3.2 x 10(-9) to 3.2 x 10(-6) mol l(-1) with the limit of detections (3sigma) being 1.3 ng ml(-1). Favorable sensitivity of TIR-RLS technique was demonstrated superior to that of high-performance liquid-chromatography (HPLC) method. The feasibility of the proposed technique was validated by the satisfactory performance of intra-assay and inter-assay BE in tablets.
This work describes the development of a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for a highly toxic impurity, FMTP (4-(4-fluorophenyl)-1-methyl-1,2,3,6-tetrahydropyridine), in paroxetine active pharmaceutical ingredient (API), followed by the subsequent validation of the methodology and transfer into a global production/quality control environment. The method was developed to achieve a detection limit of 10ppb mass fraction of FMTP in paroxetine API. An LC-MS/MS method was chosen because it provided the required sensitivity and selectivity with minimal sample preparation. This paper discusses the issues with transferring such complex methodology to a production environment. Linearity, repeatability and reproducibility of the method were demonstrated. This work shows that it is possible using the same approach that would be used for the transfer of any analytical method from R&D to a manufacturing environment.
The solutions of nine alpha, omega-bis(3-alkyl(aryl)-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl) alkanes were titrated with tetrabutylammoniumhydroxide (TBAH) in methanol, using potentiometric methods. The half neutralization potentials values were found for all cases. Potentiometric titration curves of compounds in methanol with 0.03 M TBAH are similar to those of weak acids obtained in aqueous media with strong bases. Methanol is found to be a suitable medium for the weakly acidic compounds titrated since they are poorly dissolved in other organic solvents. A comparison among the compounds having the same alkyl chains between the two ring systems has shown that basicity increases and acidity decreases as the size of alkyl chains increases. However, the compound with a substituted phenyl group was found to be the most acidic one among the examined compounds indicating that phenyl group donates ring electrons less effectively to the system. This can be attributed to the stability of the benzene ring.