Tailoring Two Chemometric Models for Determination of Three Neuromuscular Combined Medications and Application to Spiked Human Plasma.

BACKGROUND: Prednisolone, 6-mercaptopurine, and pyridostigmine bromide are co-administered together to treat a neuromuscular autoimmune disease called myasthenia gravis. Prednisolone and 6-mercaptopurine are immunosuppressant drugs. 6-Mercaptopurine is the active form of the pro-drug azathioprine. Pyridostigmine bromide is a cholinesterase enzyme inhibitor. Curently, green characteristics are taken into account by analysts when they develop new methods. OBJECTIVE: The spectra of the three compounds and plasma are highly overlapped, so this study aims to remove this overlap and determine the three components quantitatively in raw powders and spiked human plasma using green methods. METHODS: Two multivariate updated chemometric models called principle component analysis and partial least-squares were developed. Three greenness assessment tools: the eco-scale, the analytical greenness metric approach, and the green analytical method index, were used to evaluate the greenness behavior of the generated models. RESULTS: The two models were verified in accordance with Food and Drug Administration requirements, and the results were within acceptable limits. In addition they are relatively green in accordance with the abovementioned greenness evaluation tools. CONCLUSION: The developed models succeeded in determining the proposed drugs in their tertiary combinations and spiked human plasma with satisfactory precision, accuracy, and good greenness behavior. HIGHLIGHTS: Two ecologically evaluated, multivariate spectrophotometric methods were developed for the determination of pyridostigmine bromide, 6-mercaptopurine, and prednisolone in spiked human plasma.

Orthogonal projection to latent structures and first derivative for manipulation of PLSR and SVR chemometric models' prediction: A case study.

Novel manipulations of the well-established multivariate calibration models namely; partial least square regression (PLSR) and support vector regression (SVR) are introduced in the presented comparative study. Two preprocessing methods comprising first derivatization and orthogonal projection to latent structures (OPLS) are implemented prior to modeling with PLSR and SVR. Quantitative determination of pyridostigmine bromide (PR) in existence of its two associated substances; impurity a (IMP A) and impurity b (IMP B); was utilized as a case study for achieving comparison. A series consisting of 16 mixtures with numerous percentages of the studied compounds was applied for implementation of a 3 factor 4 level experimental design. Additionally, a series consisting of 9 mixtures was employed in an independent test to verify the predictive power of the suggested models. Significant improvement of predictive abilities of the two studied chemometric models was attained via implementation of OPLS processing method. The root mean square error of prediction RMSEP for the test set mixtures was employed as a key comparison tool. About PLSR model, RMSEP was found 0.5283 without preprocessing method, 1.1750 when first derivative data was used and 0.2890 when OPLS preprocessing method was applied. With regard to SVR model, RMSEP was found 0.2173 without preprocessing method, 0.3516 when first derivative data was used and 0.1819 when OPLS preprocessing method was applied.

Green Simultaneous Chromatographic Separation of Pyridostigmine Bromide and Its Related Substances in Pure Form, Tablets and Spiked Human Plasma.

A green, accurate and specific high-performance thin-layer chromatographic (HPTLC) method was developed and validated for simultaneous quantitative determination of pyridostigmine bromide (PR), impurity B (IMP B);3-hydroxy-N-methylpyridinium bromide and impurity A (IMP A); pyridin-3-yl-dimethylcarbamate. The two pharmacopeial impurities are also its main inactive metabolites. Furthermore, IMP B is known to be its alkaline-induced degradation product. Achievable separation of the studied components required silica gel HPTLC F254 plates as a stationary phase and acetone: acetic acid (80:20, v/v) as a developing system. Scanning of the separated bands was done at 260 nm. According to green solvent selection guidelines, acetone and acetic acid are eco-friendly solvents. Validation of the developed method was insured by its acquiesce to international conference on harmonization (ICH) guidelines. The introduced method was successfully achieved for the quantitative determination of PR, IMP B and IMP A in the range of 0.4-10, 2-11 and 0.4-3.5 µg/band, respectively. Successful application of the developed method was done for determination of PR in human plasma in the range of 0.6-10 µg/band, so the proposed HPTLC can be applied in the pharmacokinetic studies. The studied drug was also analyzed in Mestinon® tablets using the developed method.

Novel taste-masked orally disintegrating tablets for a highly soluble drug with an extremely bitter taste: design rationale and evaluation.

The purpose of this study was to evaluate the taste masking potential of novel solid dispersions (SDs) using Eudragit® EPO as the excipient when incorporated into the orally disintegrating tablets (ODTs) for delivering a highly soluble drug with an extremely bitter taste. The pyridostigmine bromides (PB) SDs (PBSDs) were prepared by solvent evaporation-deposition method. The physicochemical properties of PBSDs were investigated by means of differential scanning calorimetry and Fourier transformed infrared spectroscopy. The dissolution test showed that only about 8% of PB was released from PBSDs in the simulated salivary fluid in 30 s. Therefore, PBSDs were considered taste-masked and selected for formulation of PBODTs. A central composite design was employed for process optimization. Multiple linear regression analysis for process optimization revealed that the optimal PBODTs were obtained, when the microcrystalline cellulose and crospovidone were 17.16 and 5.55 (%, w/w), respectively, and the average in vivo disintegration time was 25 s. The bitterness threshold of PB was examined by a sensory test, and the threshold value was set as 3 mg in each tablet. Taste evaluation of PBODTs in 18 volunteers revealed considerable taste masking with bitterness below the threshold value. PBODTs also revealed rapid drug release (around 99%, 2 min) in the simulated gastric fluid. The mean PB plasma concentration-time profiles of PBODTs and that of the commercial tablets were comparable, with closely similar pattern. Bioequivalence assessment results demonstrated that PBODTs and the commercial tablets were bioequivalent. In conclusion, PBODTs are prepared successfully, with taste masking and rapid disintegration in the oral cavity.

Membrane electrodes for the determination of pyridostigmine bromide.

Two pyridostigmine bromide (PB) selective electrodes were investigated with 2-nitrophenyl octyl ether as a plasticizer in a polymeric matrix of carboxylated polyvinyl chloride (PVC-COOH), based on the interaction between the drug solution and the dissociated COOH groups in the PVC-COOH. One of the sensors was fabricated by using PVC-COOH only as anionic site without incorporation of an ionophore (sensor 1). The second sensor was constructed by using 2-hydroxy propyl beta-cyclodextrin as an ionophore (sensor 2). Linear responses of PB within a concentration range of 10(-3)-10(-2) and 10(-5)-10(-2) M, with slopes of 51.9 +/- 0.31 and 56.7 +/- 0.40 mV/decade over pH range of 5-10 were obtained using sensors 2 and 1, respectively. The proposed method displayed useful analytical characteristics for determination of PB in tablets with average recoveries of 100.22 +/- 0.62, and 100.15 +/- 0.72, and in plasma with average recoveries of 99.14 +/- 1.19 and 99.79 +/- 0.72, for sensors 2 and 1, respectively. The utility of 2-hydroxy propyl beta-cyclodextrin as an ionophore has a significant influence on increasing both membrane sensitivity and selectivity of sensor 2 in comparison with sensor 1. The methods were also used to determine the intact drug in the presence of its degradate, and thus could be used as stability-indicating methods. The results obtained by the proposed procedures were statistically analyzed and compared with those obtained by the U.S. Pharmacopeia method. No significant difference for either accuracy or precision was observed.

Evaluación de la legibilidad y calidad de la información suministrada en el material de acondicionado del elaborado piridostigmina DEF / Quality of legible information for the supplied conditioned material brought with the «Piridostigmina DEF» product

Antecedentes y objetivos: La utilización segura del preparado Bromuro de Piridostigmina 30 mg comprimidos DEF, para la indicación terapéutica: «Pretratamiento frente a exposición a agentes neurotóxicos inhibidores del enzima acetil colinesterasa», estará determinada por la información proporcionada al usuario del medicamento. Se procede a la confección del texto que debe acompañar al medicamento, conforme a las directrices establecidas por la Agencia Española del Medicamento y Productos Sanitarios (AEMPS) y se evalúa la calidad de esta información, considerando las siguientes circunstancias de empleo: Probable situación de estrés, facultades cognoscitivas e intelectivas deterioradas, dificultad de comprensión del término «pretratamiento», confusión con concepto «antídoto», elevadas posibilidades de ingestión masiva de comprimidos ante la idea «a mayor ingestión mayor protección» Lugar de realización: Centro Militar de Farmacia de la Defensa. – Burgos. Diseño: Revisión de las directrices vigentes en materia de información para el correcto uso de medicamentos. Análisis de riesgos derivados de las condiciones particulares de empleo de este elaborado. Confección de la información proporcionada al usuario. Elaboración de un cuestionario conforme a los criterios establecidos por el anexo 2 de la circular 2/2000 de la AEMPS, de acuerdo a la información proporcionada por el análisis de riesgos. Material y métodos: La información propuesta fue valorada mediante una encuesta realizada sobre una población de 56 personas ajenas a las profesiones sanitarias. La información proporcionada, para responder a una pregunta, es legible cuando al menos el 80 % de las respuestas son las adecuadas. Se establecen criterios de valoración de las posibles respuestas erróneas proporcionadas por los encuestados, que permitirán realizar modificaciones del texto. Resultados: Más del 90% de la población encuestada comprende la información proporcionada por el texto que acompaña a los comprimidos de Bromuro de Piridostigmina 30 mg. Ninguna pregunta presenta índices de respuestas correctas inferiores al 80%. Conclusiones: La información proporcionada resulta legible, adecuada y comprensible para los diversos tipos de usuarios del bromuro de piridostigmina comprimidos de 30 mg, para la indicación terapéutica: «pretratamiento frente la exposición a agentes neurotóxicos» (AU)

Background and Objectives: The safety use of the elaborated product Bromuro de Piridostigmina 30 mg comprimidos DEF, for the therapeutic indication: «Pre-treatment against exposition to neurotoxic agents inhibitors of the acetylcholinesterase enzyme», will be determined by the information supplied to the drug user. The text is realized to include in the container with the drug, following the directives established by the Agencia Española del Medicamento y Productos Sanitarios (AEMPS) evaluating the quality of such information, according to the considered circumstances for employment Probably stress, damage of intellective and cognitive faculties, difficulty for comprehension of the term «pre-treatment», confounding with de concept «antidote», great possibilities of massive intake of pills in the thought that «when more ingestion, more protection». Development place: Centro Militar de Farmacia de la Defensa – Burgos. Design: Check up the current directives about information for the proper use of medicines. Analysis of risks derived from the particular conditions of use in this product. Script and design the information for users. Perform a test with the established criteria (Circular 2/2000, Anexo 2, AEMPS), according to the information obtained by the risk analysis. Methods: The proposed information was evaluated by a test passes to 56 potential users, not health professionals. The information tested, answering a question, was comprehensive at least when the 80% of the questions tested were the proper ones. For the wrong answers were established criteria for evaluation, that will lead us to modify the needed parts of the text. Results: More than 90% of our population understood well the information supplied with the product. No question presented wrong answers over 80%. Conclusions: The supplied information results comprehensive, adequate and legible into the different kinds of users of the product for the indication tested (AU)

Determination of pyridostigmine bromide and its metabolites in biological samples.

Pyridostigmine bromide (PB) is a quartenary ammonium compound that inhibits the hydrolysis of acetylcholine by competitive reversible binding to acetylcholinesterase. PB is used for the symptomatic treatment of myasthenia gravis and has been applied as a prophylaxis against nerve agents. Many studies on PB have involved the reliance on techniques that extract and quantify PB in biological samples. This article presents an overview of the currently applied methodologies for the determination of PB and its metabolites in various biological samples. Articles published from January 1975 to the July 2005 were taken into consideration for the discussion of the metabolism and analytical method of PB. HPLC and GC methods have been used and discussed in most of the references cited in this review. Other methods such as RIA and CE that have been recently reported are also mentioned in this article. Basic information about the type of sample used for analysis, sample preparation, chromatographic column, mobile phase, detection mode and validation data are summarized in a table.

Determination of physostigmine and pyridostigmine in pharmaceutical formulations by capillary electrophoresis.

Physostigmine (PHY) and pyridostigmine (PYR) are two important anticholinesterase compounds with several clinical uses. Recently, PHY has been investigated for the treatment of senile dementia in Alzheimer's disease. However, both PHY and PYR have gained importance as antidotes for anticholinergic drugs. In military medicine, PYR is used as a prophylactic against nerve gas poisoning and was used in Saudi Arabia during the Gulf War in 1991. A new capillary zone electrophoresis (CZE) method for the rapid determination of PHY and PYR in pharmaceutical preparations has been developed. An untreated fused-silica capillary tube (75 microm i.d., 44 cm total length, 36.5 cm length to the detector) was employed with detection at 200 and 270 nm for PHY and PYR, respectively. The optimal separation conditions for PHY were: 50 mM boric acid-HCl buffer (pH 3.25) with 30 mM NaClO4, electrokinetic injection for 5 sec at -5 kV, temperature 25 degrees C, and separation voltage 15 kV. The optimal separation conditions for PYR were: 20 mM phosphate buffer (pH 7), electrokinetic injection for 20 sec at -10 kV, temperature 25 degrees C, and separation voltage 15 kV. The limits of detection (LOD, S/N = 3) were 70 and 60 ppb for PHY and PYR, respectively. The method can be used for the monitoring of possible main degradation products in tablets of military antidote formulations.

A validated HPLC method for the determination of pyridostigmine bromide, acetaminophen, acetylsalicylic acid and caffeine in rat plasma and urine.

A method was developed for the separation and quantification of the anti-nerve agent pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide), the analgesic drugs acetaminophen and acetylsalicylic acid, and the stimulant caffeine (3,7-dihydro-1,3,7-trimethyl-1-H-purine-2,6-dione) in rat plasma and urine. The compounds were extracted using C(18) Sep-Pak(R) cartridges then analyzed by high performance liquid chromatography (HPLC) with reversed phase C18 column, and UV detection at 280 nm. The compounds were separated using gradient of 1-85% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.5 ml/min in a period of 14 min. The retention times ranged from 8.8 to 11.5 min. The limits of detection were ranged between 100 and 200 ng/ml, while limits of quantitation were 150-200 ng/ml. Average percentage recovery of five spiked plasma samples were 70.9+/-9.5, 73.7+/-9.8, 88.6+/-9.3, 83.9+/-7.8, and from urine 69.1+/-8.5, 74.5+/-8.7, 85.9+/-9.8, 83.2+/-9.3, for pyridostigmine bromide, acetaminophen, acetylsalicylic acid and caffeine, respectively. The relationship between peak areas and concentration was linear over range between 100 and 1000 ng/ml. The resulting chromatograms showed no interfering peaks from endogenous plasma or urine components. This method was applied to analyze these compounds following oral administration in rats.

Determination of depleted uranium, pyridostigmine bromide and its metabolite in plasma and urine following combined administration in rats.

A simple and reliable method was developed for the quantification of depleted uranium, the anti nerve agent drug pyridostigmine bromide (PB;3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) and its metabolite N-methyl-3-hydroxypyridinium bromide in rat plasma and urine. The method involved using solid phase extraction and spectrophotometric determination of uranium, and high performance liquid chromatography (HPLC) with reversed phase C(18) column, and UV detection at 280 nm for PB and its metabolite. Uranium was derivatized using dibenzoylmethane (DBM) then the absorbance was measured at 405 nm. PB and its metabolite were separated using a gradient of 1--40% acetonitrile in 0.1% triflouroacetic acid water solution (pH 3.2) at a flow rate of 0.8 ml/min in a period of 14 min. Limits of detection were 2 ng/ml for uranium and 50 ng/ml for PB and its metabolite. Limits of quantitation were between 10 and 100 ng/ml for uranium and the other two analytes, respectively. Average percentage recovery of five spiked plasma samples were 83.7+/-8.6, 76.8+/-6.7, 79.1+/-7.1, and from urine 82.7+/-8.6, 79.3+/-9.5 and 78.0+/-6.2, for depleted uranium, PB and N-methyl-3-hydroxypyridinium bromide, respectively. The relationship between peak areas and concentration was linear for standards between 100 and 1000 ng/ml for all three analytes. This method was applied to analyze the above chemicals and metabolites following combined administration in rats.

Chromatographic method for the determination of diazepam, pyridostigmine bromide, and their metabolites in rat plasma and urine.

This study describes a chromatographic method for the determination of diazepam, an anxiolytic drug that is also used as an antidote against nerve agent seizures, its metabolites N-desmethyldiazepam, and temazepam, the anti-nerve agent drug pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) and its metabolite N-methyl-3-hydroxypyridinium bromide in rat plasma and urine. The compounds were extracted using C18 Sep-Pak Vac 3cc (500 mg) cartridges and separated using isocratic mobile phase of methanol, acetonitrile and water (pH 3.2) (10:40:50) at a flow-rate of 0.5 ml/min in a period of 12 min, and UV detection ranging between 240 and 280 nm. The limits of detection for all analytes ranged between 20 and 50 ng/ml, while limits of quantitation were 100 ng/ml. Average percentage extraction recoveries of five spiked plasma samples were 79.1+/-7.7, 83.5+/-6.4, 83.9+/-5.9, 71.3+/-6.0 and 77.7+/-5.6, and from urine 79.4+/-7.9, 83.1+/-6.9, 73.6+/-7.7, 74.3+/-7.1 and 77.6+/-5.9 for diazepam, N-desmethyldiazepam, temazepam, pyridostigmine bromide, and N-methyl-3-hydroxypyridinium bromide, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 1000 ng/ml. This method was applied to determine the above analytes following a single oral administration in rats as a tool to study the pharmacokinetic profile of each compound, alone and in combination.

Development of a high-performance liquid chromatographic method for the quantification of chlorpyrifos, pyridostigmine bromide, N,N-diethyl-m-toluamide and their metabolites in rat plasma and urine.

A method was developed for the separation and quantification of the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphorothioate), its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphate) and TCP (3,5,6-trichloro-2-pyridinol), the anti-nerve agent drug pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide), its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), and its metabolites m-toluamide and m-toluic acid in rat plasma and urine. The method is based on using solid-phase extraction and high-performance liquid chromatography (HPLC) with reversed-phase C18 column, and gradient UV detection ranging between 210 and 280 nm. The compounds were separated using a gradient of 1-85% acetonitrile in water (pH 3.20) at a flow-rate ranging between 1 and 1.7 ml/min over a period of 15 min. The retention times ranged from 5.4 to 13.2 min. The limits of detection ranged between 20 and 150 ng/ml, while the limits of quantitation were between 150 and 200 ng/ml. Average percentage recovery of five spiked plasma samples was 80.2+/-7.9, 74.9+/-8.5, 81.7+/-6.9, 73.1+/-7.8, 74.3+/-8.3, 80.8+/-6.6, 81.6+/-7.3 and 81.4+/-6.5, and from urine 79.4+/-6.9, 77.8+/-8.4, 83.3+/-6.6, 72.8+/-9.0, 76.3+/-7.7, 83.4+/-7.9, 81.6+/-7.9 and 81.8+/-6.8 for chlorpyrifos, chlorpyrifos-oxon, TCP, pyridostigmine bromide, N-methyl-3-hydroxypyridinium bromide, DEET, m-toluamide and m-toluic acid, respectively. The relationship between peak areas and concentration was linear over a range between 200 and 2000 ng/ml.

Determination of compounds with anticholinesterase activity in commercial drugs by a new enzyme sensor.

A suitable enzyme sensor for the analysis of anticholinesterase compounds of pharmaceutical interest is described. It is based on the competitive inhibiting properties of these compounds on the enzyme butyrylcholinesterase and it is constituted by a hydrogen peroxide amperometric electrode modified by a superimposed Nylon membrane containing two chemically immobilized biological mediators (butyrylcholinesterase and choline oxidase). Some applications to the analysis of several pharmaceutical forms containing different compounds showing anticholinesterase activity are also reported and evaluated.

Radioimmunoassay of pyridostigmine in plasma and tissues.

A sensitive and specific radioimmunoassay is reported for the cholinergic agent, pyridostigmine. Antibodies were raised in rabbits against a conjugate of pyridostigmine and bovine serum albumin. The assay can detect as little as 2.5 ng/ml of drug directly in plasma and tissue homogenates. Structurally similar compounds and major metabolites are not recognized by the antibody. The specificity of the antibody has been confirmed by utilizing high pressure liquid chromatography. Plasma concentration-time profiles and tissue distribution of the drug were determined by this method in rat after intramuscular administration of pyridostigmine.

Reversed-phase, ion-pair liquid chromatography of quaternary ammonium compounds: determination of pyridostigmine, neostigmine and edrophionium in biological fluids.

A reversed-phase, ion-pair liquid chromatographic method for the quantitative determination of quaternary acetylcholinesterase inhibitors is described. The method uses an ion-pair extraction to isolate the drugs from biological material prior to liquid chromatographic separation and online UV detection at 214 nm. Quantiation down to 5 ng/ml and within-day precison with coefficient of variation (C.V.) of 1.5% (n = 10, x = 100 ng/ml) for neostigmine, C.V., 1.7% (n = 10, x = 80 ng/ml) for pyridostigmine and C.V., 1.5% (n = 10, x = 100 ng/ml) for edrophonium have been achieved. The assay was designed for pharmacokinetic studies of these drugs in anesthetized patients.

The excretion and metabolism of oral 14C-pyridostigmine in the rat.

1. Pyridostigmine labelled with carbon-14 in the methyl group of the quaternary nitrogen has been used to investigate the excretion and metabolism of the drug after administration of single doses (500 mug) to the rat by stomach tube.2. Pyridostigmine is slowly excreted in the urine; the maximum excretion occurs between 1-3 hr after administration. In 24 hr 42% of the dose is excreted in urine and 38.4% is present in faeces and intestinal contents.3. The peak concentration of radioactivity in liver and blood occurs about 2 hr after administration.4. About 75% of the radioactivity in urine is present as unchanged pyridostigmine, the remainder as metabolite.5. The results are compared with those previously obtained after oral administration of neostigmine.6. It is concluded that after oral administration the absorption of pyridostigmine is greater and the metabolism substantially less than that of neostigmine.