Ultrasensitive chemiluminescence assay for cimetidine detection based on the synergistic improving effect of Au nanoclusters and graphene quantum dots.

A novel and sensitive chemiluminescence (CL) procedure based on the synergetic catalytic effects of gold nanoclusters (Au NCs) and graphene quantum dots (GQDs) was developed for the reliable measurement of cimetidine (CM). The initial experiments showed that the KMnO4 -based oxidation of alkaline rhodamine B (RhoB) generated a very weak CL emission, which was intensively enhanced in the simultaneous presence of Au NCs and GQDs. CL intermediates can be adsorbed and gathered on the surface of Au NCs, becoming more stable. GQDs participate in the energy transferring processes and facilitate them. These improving effects were simultaneously obtained by adding both Au NCs and GQDs into the RhoB-KMnO4 reaction. Consequently, the increasing effect of the Au NCs/GQDs mixture was more than that of pure Au NCs or GQDs, and a new nano-assisted powerful CL system was achieved. Furthermore, a marked quenching in the emission of the introduced CL system was observed in the presence of CM, so the system was examined to design a sensitive sensor for CM. After optimization of influencing parameters, the linear lessening in CL emission intensity of KMnO4 -RhoB-Au NCs/GQDs was verified for CM concentrations in the range 0.8-200 ng ml-1 . The limit of detection (3Sb /m) was 0.3 ng ml-1 . Despite being a simple CL method, good sensitivity was obtained for CM detection with reliable results for CM determination in human urine samples.

Development and validation of RP-HPLC method for simultaneous determination of cefpodoxime proxetil and H2 receptor antagonists in pharmaceutical dosage forms.

A new method on RP-HPLC is devised and validated, as per ICH guidelines, for the synchronous estimation of cefpodoxime proxetil and H2-receptor antagonits that are Cimetidine, Famotidine and Ranitidine. The method is simple, accurate, expeditious, reproducible, robust and precise. Chromatography was done on a C18 (250 x 4.6mm) column with methanol: water as mobile phae in the ratio of 70:30 (v/v), pumped at a flow rate of 1ml/min and pH was maintained using 85% ortho-phosphoric acid at 3. The λ max 240 nm was preferred for UV detection. A good linear relationship was attained, over the concentration ranges of 20-70 µg/ml and 5-30µg/ml, for cefpodoxime proxetil and H2 blockers respectively, with a correlation coefficient of R= 0.9987 to 0.9992. The method was validated and found precised (i.e. intra day and interday analysis) with RSD <2%. LOD and LOQ observations were under 0.4806 to 2.6069µg/ml which proved the method to be sensitive. The method provided satisfactory results of robustness and reproducibility, when validated and applied successfully for analysis of dosage forms.

A quantitative HPLC-MS/MS method for studying internal concentrations and toxicokinetics of 34 polar analytes in zebrafish (Danio rerio) embryos.

An analytical method using high-performance liquid chromatography-tandem mass spectrometry was developed to determine internal concentrations of 34 test compounds such as pharmaceuticals and pesticides in zebrafish embryos (ZFE), among them, cimetidine, 2,4-dichlorophenoxyacetic acid, metoprolol, atropine and phenytoin. For qualification and quantification, multiple reaction monitoring mode was used. The linear range extends from 0.075 ng/mL for thiacloprid and metazachlor and 7.5 ng/mL for coniine and clofibrate to 250 ng/mL for many of the test compounds. Matrix effects were strongest for nicotine, but never exceeded ±20 % for any of the developmental stages of the ZFE. Method recoveries ranged from 90 to 110 % from an analysis of nine pooled ZFE. These findings together with the simple sample preparation mean this approach is suitable for the determination of internal concentrations from only nine individual ZFE in all life stages up to 96 h post-fertilization. Exemplarily, the time course of the internal concentrations of clofibric acid, metribuzin and benzocaine in ZFE was studied over 96 h, and three different patterns were distinguished, on the basis of the speed and extent of uptake and whether or not a steady state was reached. Decreasing internal concentrations may be due to metabolism in the ZFE.

Controlled-pH tissue cleanup protocol for signal enhancement of small molecule drugs analyzed by MALDI-MS imaging.

The limit of detection of low-molecular weight compounds in tissue sections, analyzed by matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), was significantly improved by employing sample washing using a pH-controlled buffer solution. The pH of the washing solutions were set at values whereby the target analytes would have low solubility. Washing the tissue sections in the buffered solution resulted in removal of endogenous soluble ionization-suppressing compounds and salts, while the target compound remained in situ with minor or no delocalization during the buffered washing procedure. Two pharmaceutical compounds (cimetidine and imipramine) and one new protease inhibitor compound were successfully used to evaluate the feasibility of the pH-controlled tissue washing protocol for MALDI-MSI. Enhancement in signal-to-noise ratio was achieved by a factor of up to 10.

Determination of ranitidine, nizatidine, and cimetidine by a sensitive fluorescent probe.

A validated, simple, and sensitive fluorescence quenching method for the determination of ranitidine, nizatidine, and cimetidine in tablets and biological fluids is presented. This is the first single fluorescence method reported for the analysis of all three H(2) antagonists. The competitive reaction between the investigated drug and the palmatine probe for the occupancy of the cucurbit[7]uril (CB[7]) cavity was studied using spectrofluorometry. CB[7] was found to react with the probe to form a stable complex. The fluorescence intensity of the complex was also enhanced greatly. However, the addition of the drug dramatically quenched the fluorescence intensity of the complex. Accordingly, a new fluorescence quenching method for the determination of the studied drugs was established. The different experimental parameters affecting the fluorescence quenching intensity were studied carefully. At optimum reaction conditions, the rectilinear calibration graphs between the fluorescence quenching values (ΔF) and the medicament concentration were obtained in the concentration range of 0.04-1.9 µg mL(-1) for the investigated drugs. The limits of detection ranged from 0.013 to 0.030 µg mL(-1) at 495 nm using an excitation wavelength of 343 nm. The proposed method can be used for the determination of the three H(2) antagonists in raw materials, dosage forms and biological fluids.

Enantioselective in-line and off-line CE methods for the kinetic study on cimetidine and its chiral metabolites with reference to flavin-containing monooxygenase genetic isoforms.

An in-line screening and an off-line chiral CE method were developed to determine the stereoselectivity of flavin-containing monooxygenase (FMO) isoforms using cimetidine (CIM) as a substrate. The S-oxygenation of CIM was investigated using achiral chemical oxidants and (human supersomes) enzymatic metabolism procedures. In the off-line setup, the chiral selector sulfobutylether-beta-CD was chosen to separate the CIM S-oxide (CSO) metabolites. The electrophoretic migration order of CSO was confirmed to be (+) before (-) through the use of single enantiomers obtained by preparative chromatography. For the electrophoretically mediated microanalysis method, the in-line enzymatic reaction was performed in 100 mM phosphate reaction buffer (pH 8.3), whereas 50 mM phosphate buffer with 30 mM chiral selector (pH 2.5) was used as a BGE. During the screening of FMO isoenzymes by the electrophoretically mediated microanalysis method, formation of the new chiral center on the CIM sulfur was found to be stereoselective. FMO1 produces more (-)-CSO-enantiomer, while FMO3 generates mainly (+)-CSO-enantiomer. On the other hand, FMO5 shows no activity. The kinetic constants of FMO1 and FMO3 were measured by the off-line method. A K(m)=4.31 mM for the formation of the (+)-CSO-enantiomer and a K(m)=4.56 mM for the (-)-CSO-enantiomer are reported for the first time for FMO1.

Colorimetric assay of cimetidine in the presence of its oxidative degradates.

Three simple, accurate, and sensitive colorimetric methods for the determination of cimetidine (Cim) in pure form, in dosage forms, and in the presence of its oxidative degradates were developed. These methods are indirect, involve the addition of excess oxidant [N-bromosuccinimide (NBS) for method A; cerric sulfate [Ce(SO4)2] for methods B and C] of known concentration in acid medium to Cim, and the determination of the unreacted oxidant by measurement of the decrease in absorbance of amaranth dye for method A, chromotrope 2R for method B, and rhodamine 6G, for method C at a suitable maximum wavelength, lambda max: 520, 528, and 525 nm, for the 3 methods, respectively. Regression analysis of the Beer plots showed good correlation in the concentration ranges of 0.2-4.4 microg/mL for method A, and 0.2-3.4 microg/mL for methods B and C. The apparent molar absorptivity, Sandell sensitivity, and detection and quantitation limits were evaluated. The stoichiometric ratio between the drug (Cim) and the oxidant (NBS or Ce4+) was estimated. The validity of the proposed methods was tested by analyzing pure and dosage forms containing Cim with relative standard deviation < or = 1.18. The proposed methods could successfully determine the studied drug with varying excess of its oxidative degradation products, with recovery between 99.2 and 101.8, 100.2 and 102.8, and 99.8 and 102.0% for methods A-C, respectively.

Spectrophotometric determination of H(2)-receptor antagonists via their oxidation with cerium(IV).

A simple, accurate and sensitive spectrophotometric method has been developed and validated for determination of H(2)-receptor antagonists: cimetidine, famotidine, nizatidine and ranitidine hydrochloride. The method was based on the oxidation of these drugs with cerium(IV) in presence of perchloric acid and subsequent measurement of the excess Ce(IV) by its reaction with p-dimethylaminobenzaldehyde to give a red colored product (lambda(max) at 464nm). The decrease in the absorption intensity of the colored product (DeltaA), due to the presence of the drug was correlated with its concentration in the sample solution. Different variables affecting the reaction were carefully studied and optimized. Under the optimum conditions, linear relationships with good correlation coefficients (0.9990-0.9994) were found between DeltaA values and the concentrations of the drugs in a concentration range of 1-20microgml(-1). The assay limits of detection and quantitation were 0.18-0.60 and 0.54-1.53microgml(-1), respectively. The method was validated, in terms of accuracy, precision, ruggedness and robustness; the results were satisfactory. The proposed method was successfully applied to the determination of the investigated drugs in pure and pharmaceutical dosage forms (recovery was 98.3-102.6+/-0.57-1.90%) without interference from the common excipients. The results obtained by the proposed method were comparable with those obtained by the official methods.

Assessing the Detection Limit of a Minority Solid-State Form of a Pharmaceutical by 1H Double-Quantum Magic-Angle Spinning Nuclear Magnetic Resonance Spectroscopy.

The lower detection limit for 2 distinct crystalline phases by 1H magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) is investigated for a minority amount of cimetidine (anhydrous polymorph A) in a physical mixture with the anhydrous HCl salt of cimetidine. Specifically, 2-dimensional 1H double-quantum (DQ) MAS NMR spectra of polymorph A and the anhydrous HCl salt constitute fingerprints for the presence of each of these solid forms. For solid-state NMR data recorded at a 1H Larmor frequency of 850 MHz and a MAS frequency of 30 kHz on ∼10 mg of sample, it is shown that, by following the pair of cross-peaks at a 1H DQ frequency of 7.4 + 11.6 = 19.0 ppm that are unique to polymorph A, the level of detection for polymorph A in a physical mixture with the anhydrous HCl salt is a concentration of 1% w/w.

Qualitative and simultaneous quantitative analysis of cimetidine polymorphs by ultraviolet-visible and shortwave near-infrared diffuse reflectance spectroscopy and multivariate calibration models.

The object of the present study was to investigate the feasibility of applying ultraviolet-visible and shortwave near-infrared diffuse reflectance spectroscopy (UV-vis-SWNIR DRS) coupled with chemometrics in qualitative and simultaneous quantitative analysis of drug polymorphs, using cimetidine as a model drug. Three polymorphic forms (A, B and D) and a mixed crystal (M1) of cimetidine, obtained by preparation under different crystallization conditions, were characterized by microscopy, X-ray powder diffraction (XRPD) and infrared spectroscopy (IR). The discriminant models of four forms (A, B, D and M1) were established by discriminant partial least squares (PLS-DA) using different pretreated spectra. The R and RMSEP of samples in the prediction set by discriminant model with original spectra were 0.9959 and 0.1004. Among the quantitative models of binary mixtures (A and D) established by partial least squares (PLS) and least squares-support vector machine (LS-SVM) with different pretreated spectra, the LS-SVM models based on original and MSC spectra had better prediction effect with a R of 1.0000 and a RMSEP of 0.0134 for form A, and a R of 1.0000 and a RMSEP of 0.0024 for form D. For ternary mixtures, the established PLS quantitative models based on normalized spectra had relatively better prediction effect for forms A, B and D with R of 0.9901, 0.9820 and 0.9794 and RMSEP of 0.0471, 0.0529 and 0.0594, respectively. This research indicated that UV-vis-SWNIR DRS can be used as a simple, rapid, nondestructive qualitative and quantitative method for the analysis of drug polymorphs.

Active transport of cimetidine into human milk.

Most xenobiotics are transferred from blood into breast milk by passive diffusion. However, an active transport mechanism has been speculated for cimetidine, and the purpose of this study was to characterize cimetidine transfer into human milk. Twelve healthy lactating volunteers received single oral doses of 100, 600, and 1200 mg cimetidine in a randomized, crossover design on 3 different days. Blood and milk specimens were collected and assayed for cimetidine. In vitro measurements, including skim to whole milk concentration ratio, milk pH, and free fractions in serum and milk were used for a diffusion model prediction of milk to serum concentration ratio of cimetidine; the mean milk/serum ratio (+/- SD) was 1.05 +/- 0.18. The observed milk/serum ratio (5.77 +/- 1.24) was 5.5 times higher than the milk/serum ratio predicted by diffusion. The observed milk/serum ratio for the three dosing regimens were not significantly different from one another. Time of peak concentration (tmax) in milk (3.3 +/- 0.7 hours) displayed a delay compared with serum tmax (1.7 +/- 0.6 hours). Oral clearance for 1200 mg cimetidine dose (0.47 +/- 0.11 L/hr/kg) was significantly lower compared with oral clearance values for 100 and 600 mg cimetidine doses (0.59 +/- 0.11 and 0.57 +/- 0.13 L/hr/kg, respectively). The maternal dose of cimetidine ingested by a suckling infant based on body weight was estimated to be 6.7%, which appears to be safe under normal conditions. This study provides the first definitive evidence of an active transport system for drug transfer into human milk, which may have broader consequences for the suckling infant.

The use of on-line and off-line chromatographic extraction techniques coupled with mass spectrometry for support of in vivo and in vitro assays in drug discovery.

We have investigated various sample chromatographic extraction and sample preparation methods for liquid chromatography mass spectrometry analysis in order to increase the throughput of various in vivo and in vitro assays in support of drug discovery. The results indicated that direct plasma injection, although certainly faster than conventional protein precipitation for sample preparation, had problems associated with column longevity and overall robustness. Frequently a single study could not be completed without column replacement. On-line solid phase extraction, on the other hand, compared well with off-line solid phase extraction, using our LC extraction column design, as contamination of the extraction column was minimized by back flushing using the Gilson syringe pump. Finally, on-line solid phase extraction for support of Caco-2 permeability studies worked very well for both single components and mixtures as the matrix was much simpler, presenting fewer contamination problems.

(Imidazolylphenyl)formamidines. A structurally novel class of potent histamine H2 receptor antagonists.

Structure-activity considerations of N alpha-guanylhistamine, the first compound found with detectable H2-antagonist activity, led to the synthesis of a series of conformationally rigid guanylhistamine analogues, namely, (imidazolylphenyl)guanidines, imidazolylbenzamidines, and (imidazolylphenyl)formamidines. It was found that in the guanidine and benzamidine classes, the meta-substituted derivatives (3, 4, 7, and 8) possessed H2-antagonist activity, whereas in the class of formamidines, only the para-substituted derivative 10 was found active. A subsequent increase in the size of the substituent at the formamidino group of 10 led to compounds (15-20) of high H2-antagonist affinity, which was related to the gastric antisecretory effect. Members of this structurally novel class of H2 antagonists were 20- to 50-fold more potent than cimetidine both "in vitro" and "in vivo". Structure-activity relationships are discussed in terms of ionization properties, partitioning behavior, conformational aspects of the selected compound 17, and of possible modes of interaction with the histamine H2 receptor. It was found that the formamidine moiety was an important structural feature and that H2-antagonist activity requires correct steric and electronic properties. Compound 17 (DA 4577), owing to its pharmacological profile and demonstrated safety in animals, was selected to be clinically investigated.

Intestinal clearance of H2-antagonists.

Jejunal perfusion of cimetidine resulted in the appearance of lumenal cimetidine sulfoxide in both rats and humans. In the rat, ileal perfusion yielded negligible sulfoxide metabolite as compared with that of the jejunum. Jejunal co-perfusion of an anionic-exchange inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, blocked the appearance of drug sulfoxide, and methionine co-perfusion yielded concentration-dependent inhibition of lumenal cimetidine sulfoxide. Intravenous injection of high concentrations of cimetidine sulfoxide did not produce detectable lumenal metabolite levels during jejunal perfusion of drug-free buffer, providing in situ evidence that lumenal metabolite is generated by the small intestine. The extent of the appearance of lumenal sulfoxide was significantly greater for cimetidine than for the other three marketed H2-antagonists in rat jejunum. Variable intestinal clearance of this extensively prescribed class of therapeutic agents may contribute to their absorption variability.

High-pressure liquid chromatographic determination of cimetidine in plasma and urine.

An assay is described for the determination of the H2-receptor antagonist, cimetidine, in human plasma and urine. Alkalinized plasma or urine was extracted with methylene chloride, the organic phase was evaporated, and the reconstituted residue was analysed by high-pressure liquid chromatography (HPLC) using a reversed-phase prepacked plastic column housed in a radial compression module. The metabolite, cimetidine sulfoxide, was identified but could not be quantitated due to interference from the solvent front. The sensitivity limit of the assay was 25 ng/ml. The assay was applied to the measurement of plasma and urine samples in a pilot pharmacokinetic study. Cimetidine was substantially absorbed and rapidly eliminated (plasma elimination half-life of 112-130 min). Plasma cimetidine concentrations could be measured to 12 hr after a 200-mg dose (iv or oral), but they were below the sensitivity of the assay by 24 hr. Urinary excretion of unmetabolized cimetidine accounted for 40-50% of the administered dose in the first 12 hr. This assay is simpler and more sensitive than those previously described, and it is suitable for the measurement of cimetidine in plasma and urine of subjects receiving doses appropriate for clinical use.

Ion-selective electrodes for the H2-receptor antagonists cimetidine and ranitidine.

Liquid-membrane and polyvinyl chloride (PVC)-matrix ion-selective electrodes (ISE) that respond to the cationic forms of cimetidine and ranitidine are described. The ion-exchangers were the salts of cimetidine and ranitidine with tetrakis(m-chlorophenyl)borate dissolved in p-nitrocumene or entrapped in PVC polymer in the presence of 2-nitrophenyl octyl ether as plasticizer. The electrodes exhibited a near-Nernstian response in the range 10(-2)-10(-6)M (working pH range 2-7) for ranitidine, and 10(-2)-2 X 10(-5)M (pH 2-6) for cimetidine. Very small PVC-matrix ISE with internal diameters as small as 0.035 inches were constructed and used in combination with small cuvettes, so that measurements could be carried out in 250 muL of stirred solution. The electrodes were applied successfully for the determination of the pKa of the protonated bases and for the determination of the drugs in pharmaceutical preparations. New selective and effective solid-state extraction procedures are described for the extraction of ranitidine from urine and serum samples. Potentiometric methods were developed for the determination of ranitidine in urine and serum samples during a pharmacokinetic experiment.

Preparation of a cimetidine ion-selective electrode and its application to pharmaceutical analysis.

A novel cimetidine ion-selective electrode is prepared, characterized and used in pharmaceutical analysis. The electrode incorporates PVC-membrane with cimetidine-phospohotungstate ion pair complex. The electrode exhibits a Nernstian response for cimetidine in the concentration range 1.0 x 10(-5)-1.0 x 10(-2) M with a slope of 58+/-1 mV per decade. The limit of detection is 5.0 x 10(-6) M. The electrode displays a good selectivity for cimetidine with respect to a number of common foreign inorganic and organic species. It can be used in the pH range 3.0-5.5. The membrane sensor was successfully applied to the determination of cimetidine in its tablets as well as its recovery from a urine sample.

Spectrophotometric determination of cimetidine in the presence of its acid-induced degradation products.

Cimetidine has been determined in the presence of its acid-induced degradation products using a second derivative (D2-) spectrophotometric method (method I) or a colorimetric method (method II). The former is based on D2-value measurement at 216 nm, whilst the latter depends on charge-transfer complexation with dichlorophenol-indophenol. The two methods are proved to be stability indicating, since plots of log C% versus time were linear. The application to cimetidine determination in tablets and ampoules gave good results.

Nondestructive tablet hardness testing by near-infrared spectroscopy: a new and robust spectral best-fit algorithm.

A new algorithm using common statistics was proposed for nondestructive near-infrared (near-IR) spectroscopic tablet hardness testing over a range of tablet potencies. The spectral features that allow near-IR tablet hardness testing were evaluated. Cimetidine tablets of 1-20% potency and 1-7 kp hardness were used for the development and testing of a new spectral best-fit algorithm for tablet hardness prediction. Actual tablet hardness values determined via a destructive diametral crushing test were used for construction of calibration models using principal component analysis/principal component regression (PCA/PCR) or the new algorithm. Both methods allowed the prediction of tablet hardness over the range of potencies studied. The spectral best-fit method compared favorably to the multivariate PCA/PCR method, but was easier to develop. The new approach offers advantages over wavelength-based regression models because the calculation of a spectral slope averages out the influence of individual spectral absorbance bands. The ability to generalize the hardness calibration over a range of potencies confirms the robust nature of the method.

Estimation of impurity profiles of drugs and related materials Part 15. Identification of minor impurities in cimetidine.

Besides several known impurities in cimetidine, two additional compounds at levels below 0.1% were detected by ion-pair reversed-phase high-performance liquid chromatography (HPLC). The impurities were isolated from crude cimetidine using normal-phase preparative HPLC. 1H and 13C NMR and mass spectrometric investigations revealed the structures of the impurities to be 2,5-bis[(N'-cyano-N"-methyl)guanidinoethylthiomethyl]-4-methylimid azole (VII) and 1,8-bis[(N'-cyano-N"-methyl)guanidino]-3,6-dithiaoctane (VIII). These structures were verified by synthesis of the impurities and comparison of the spectra and chromatographic (HPLC and TLC) retention data of the isolated and synthesized materials.