Defatted hempseed meal altered the metabolic profile of fermented yogurt and enhanced the ability to alleviate constipation in rats.

BACKGROUND: Hempseeds (Cannabis sativa L.) are rich in easily digestible proteins, fats, polyunsaturated fatty acids, and insoluble fiber and are of high nutritional value. Probiotics have been found to relieve constipation, which solves a health problem that constantly troubles a lot of people. Therefore, the changes in the metabolites of fermented yogurt with or without 10% defatted hempseed meal (10% SHY or 0% SHY respectively) were studied and their laxative effects were examined through animal experiments. RESULTS: Amino acids and peptides, terpene glycosides, carbohydrates, lineolic acids, and fatty acids were found to be the major contributors to the discrimination of the metabolic profile between 0% SHY and 10% SHY. The differentially accumulated metabolites may lead to the discrepancy in the yogurt's functionality. Animal experiments showed that the 10% SHY treatment prevented constipation by increasing feces number, fecal water content, and small intestinal transit rate and reducing inflammatory injury in loperamide-induced constipated rats. Further analysis of the gut microbiota revealed that 10% SHY gavage increased the relative abundances of the Lactobacillus, Allobaculum, Turicibacter, Oscillibacter, Ruminococcus, and Phascolarctobacterium genera in the constipated rats, whereas Akkermansia, Clostridium_XIVa, Bacteroides, Staphylococcus, and Clostridium_IV were decreased. The combination of defatted hempseed meal and probiotics was found to be effective in relieving constipation, probably due to the enriched amino acids and peptides, such as Thr-Leu and lysinoalanine through correlation analysis. CONCLUSION: Our findings indicated that defatted hempseed meal in yogurt altered the metabolic profile and effectively alleviated constipation in rats, which is a promising therapeutic candidate for constipation. © 2023 Society of Chemical Industry.

Hollow Fiber Liquid-Phase Microextraction At-Line Coupled to Capillary Electrophoresis for Direct Analysis of Human Body Fluids.

A simple and cheap all-in-one concept for at-line coupling of hollow fiber liquid-phase microextraction (HF-LPME) to commercial capillary electrophoresis (CE) is demonstrated, which enables the direct analysis of complex samples. A disposable microextraction device compatible with injection systems of Agilent CE instruments is proposed, which consists of a short segment of a porous HF attached to a tapered polypropylene holder. The holder maintains a constant position of the HF in a CE vial during extraction and simultaneously guides the injection end of a separation capillary into the HF lumen for automated CE injection and analysis. In a typical analytical procedure, the HF is impregnated with a water-immiscible solvent, its lumen is filled with 5 µL of an aqueous acceptor solution, and the microextraction device is placed in a 2 mL glass CE vial containing 550 µL of a donor solution. The vial is agitated at 750 rpm for 10 min, and the resulting acceptor solution is injected directly from the HF lumen into the commercial CE. No additional manual handling is required, except for the transfer of the CE vial to the CE autosampler. Multiple complex samples can be simultaneously pretreated in a multiple-well plate format, thus significantly reducing the total analysis time. Suitability of the analytical method is demonstrated by the direct determination of model basic drugs (nortriptyline, haloperidol, loperamide, and papaverine) in physiological solutions, urine, and dried blood spot (DBS) samples. Repeatability of the method is better than 12.8% (%RSD), extraction recoveries range between 34 and 76%, and enrichment factors are 37-84. The method is linear in a range of 2 orders of magnitude (R2 ≥ 0.9977) with limits of detection of 0.7-1.55 µg/L. The method has a high potential for the direct analysis of DBS samples since DBS elution and HF-LPME are performed simultaneously during the 10 min agitation. The manual DBS handling is thus reduced to inserting the DBS punch into the CE vial only. Moreover, the universal character of the HF-LPME might extend the applicability of the method to a wide range of analytes/matrices, and combination with other commercial detectors might improve the selectivity/sensitivity of the CE analysis.

Effect and mechanism of functional compound fruit drink on gut microbiota in constipation mice.

Compound fruit drink (CFD) is a functional drink prepared with fruit, Chinese herbs and prebiotic fructooligosaccharide as the main ingredients. Loperamide hydrochloride was used to establish a mouse model of constipation. And the effect of CFD on the improvement of constipation and the impact on gut microbiota were studied. The results showed that CFD significantly enhanced intestinal motility in constipated mice (P < 0.05). It significantly improved serum levels of gastrointestinal regulatory-related peptides, elevated the short-chain fatty acids (SCFAs) content and alleviated colonic injury. Meanwhile, CFD also up-regulated the mRNA expression levels of AQP3, AQP9, SCF and c-Kit and the related protein expression levels. Fecal microbial results showed that the CFD medium-dose group significantly increased species richness. Furthermore, CFD increased the abundance of potentially beneficial bacteria and reduced the number of potentially pathogenic bacteria. This study indicated that CFD was a promising functional drink for effectively relieving constipation.

Loperamide as a Potential Drug of Abuse and Misuse: Fatal Overdoses at the Medical University of South Carolina.

Loperamide is an over-the-counter, µ-opioid receptor agonist commonly used as an antidiarrheal agent. Loperamide was thought to have minimal abuse potential due to its low bioavailability and limited central nervous system activity; however, there have been increasing reports of loperamide misuse in supratherapeutic doses to achieve euphoria and/or avoid opioid withdrawal. A literature review suggests a rise in loperamide abuse was inevitable, with substantial increases in reported cases over the last decade. Five fatal cases of toxic medication use where loperamide was listed as a primary or contributory cause of death were identified at the Medical University of South Carolina. The characteristic autopsy demographics and findings are described, and the mechanisms of abuse and toxicity of loperamide are reviewed. Loperamide overdoses are a growing concern from both a forensic and clinical standpoint, and the frequency of reported cases will likely increase as awareness grows within the medical and toxicological communities.

Study of the physicochemical properties and stability of solid dispersions of loperamide and PEG6000 prepared by spray drying.

Solid dispersions of PEG6000 and loperamide-a poorly water-soluble agent-were prepared by spray drying. Their physicochemical properties were evaluated immediately after preparation. The dissolution was higher than that of pure crystalline loperamide. DSC- and XRD-measurements revealed that in the dispersions, loperamide is partially present in the crystalline state. A eutectic state diagram was obtained. The samples containing 20% loperamide were stored under different conditions (40 degrees C and 0% RH, 25 degrees C and 52% RH, 4 degrees C and 0% RH) to investigate their stability as a function of time. The dissolution properties deteriorate upon storage at high temperature (40 degrees C and 0% RH) and in conditions of higher relative humidity (25 degrees C and 52% RH). The DSC-curves clearly indicate an increase in the amount of crystalline compound under these conditions. From these observations it could be concluded that loperamide, which is partially crystalline and partially amorphous in the freshly prepared samples, continues to crystallize under these conditions, resulting in progressively poorer dissolution properties.

Phase behaviour analysis of solid dispersions of loperamide and two structurally related compounds with the polymers PVP-K30 and PVP-VA64.

The purpose of the present study was to investigate the influence of the structure of a poorly water soluble model drug (loperamide) on the phase behaviour in solid dispersions with PVP-K30. Dispersions with PVP-VA64, a less hydrophilic polymer, were investigated as well in order to study the influence of differences in polymer structure and water content of the samples. The solid dispersions of PVP-K30 or PVP-VA64 with loperamide as well as with two fragments of this molecule were prepared by spray drying. The amount of residual solvents and water was determined with GC and thermogravimetric analysis (TGA). The drug loading of the dispersions was determined using high performance liquid chromatography (HPLC). The solid state properties were evaluated using powder-XRD, IR-spectroscopy and MT-DSC. All mixtures containing loperamide proved to be completely amorphous, whereas the dispersions containing the fragments are only amorphous in case the polymer content is high. The phase diagrams that were constructed clearly show that loperamide exhibits a different behaviour in the solid dispersions than its two building blocks. They also point to the presence of specific intermolecular compound--polymer interactions in the dispersions of one of the fragments with the two polymers. This was confirmed by the IR-results. Despite structural similarities, interactions in dispersions containing loperamide are far less important. In dispersions containing high concentrations of the other fragment, the DSC curves give indications for polymorphism whereas IR and XRD-spectra point towards inclusion of solvent in these samples.

Spectrophotometric determination of loperamide hydrochloride by acid-dye and charge-transfer complexation methods in the presence of its degradation products.

Two simple, sensitive and accurate spectrophotometric methods for the determination of loperamide hydrochloride (lop. HCl) are described. The first method is based on the formation of ion-pair association complex (1:1) with bromothymol blue (BTB), bromophenol blue (BPB) and naphthol blue black B (NBB). The coloured products are extracted into chloroform, and measured spectrophotometrically at 414 (BTB), 415 (BPB) and 627 nm (NBB). Beer's law was obeyed in the ranges of 5-35, 5-30 and 0.8-11.2 microg ml(-1) for BTB, BPB and NBB methods, respectively. The method was found to be specific for the analysis of the drug in presence of its degradation products which can be detected by HPLC procedure. The second method is based on the reaction of the basic loperamide with iodine in chloroform to give molecular charge-transfer complex with intense bands at 295 and 363 nm. Beer's law was obeyed in the ranges 2.5-17.5 and 2.5-22.5 microg ml(-1) for the method at 295 and 363 nm, respectively. Optimization of the different experimental conditions are described for both methods. The proposed methods have been applied successfully for the analysis of the drug in pure form and in its dosage forms. The methods have the advantage of being highly sensitive and simple for the determination of a small dose drug, which is also a weak UV-absorbing compound.

Spectrofluorimetric and derivative absorption spectrophotometric techniques for the determination of loperamide hydrochloride in pharmaceutical formulations.

Simple, sensitive and rapid spectrofluorimetric and derivative absorption spectrophotometric procedures are described for the accurate determination of loperamide hydrochloride. The spectrofluorimetric method involves the measurement of the fluorescence of the compound in an ethanol-sulphuric acid mixture (90:10, v/v) using either the direct or the synchronous modes of measurement. Optimum conditions for maximum fluorescence are described. The derivative spectrophotometric method involves the measurement of either the second derivative peak amplitude (crest to trough, i.e. maximum to minimum) between 258 and 263 nm or the second derivative peak height (i.e. maximum to zero line) at 224 nm of an ethanolic solution of the drug. The proposed methods have been used for the determination of loperamide in pharmaceutical formulations. Compared to the pharmacopoeial method, the proposed methods give equally accurate (t-test) and precise (f-test) results. The proposed methods have the advantages of being highly sensitive for the determination of small concentrations of loperamide, a weak UV-absorbing compound prescribed in low doses.

LC-MS determination and bioavailability study of loperamide hydrochloride after oral administration of loperamide capsule in human volunteers.

The purpose of the present study was to develop a standard protocol for loperamide hydrochloride bioequivalence testing. For this purpose, a simple rapid and selective LC-MS method utilizing a single quadrupole mass spectrometer was developed and validated for the determination of loperamide hydrochloride in human plasma, and we followed this with a bioavailability study. Methyl tert-butylether (MTBE) was used to extract loperamide hydrochloride and ketoconazole (internal standard (IS)) from an alkaline plasma sample. LC separation was performed on a Zorbax RX C18 column (5 microm, 2.1 mm x 150 mm) using acetonitrile-water-formic acid (50:50:0.1 (v/v)) as a mobile phase. The retention times of loperamide hydrochloride and IS were 1.2 and 0.8 min, respectively. Quadrupole MS detection was by monitoring at m/z 477 (M + 1) corresponding to loperamide hydrochloride and at m/z 531 (M + 1) for IS. The described assay method showed acceptable precision, accuracy, linearity, stability, and specificity. The bioavailability of loperamide hydrochloride was evaluated in eight healthy male volunteers. The following pharmacokinetic parameters were elucidated after administering a single dose of four 2mg capsules of loperamide: the area under the plasma concentration versus time curve from time 0 to 72 h (AUC72 h) 19.26 +/- 7.79 ng h/ml; peak plasma concentration (Cmax) 1.18 +/- 0.37 ng/ml; time to Cmax (Tmax) 5.38 +/- 0.74 h; and elimination half-life (t1/2) 11.35 +/- 2.06 h. The developed method was successfully used to study the bioavailability of a low dose (8 mg) of loperamide hydrochloride.

Determination of the active ingredient loperamide hydrochloride in pharmaceutical caplets by high performance thin layer chromatography with ultraviolet absorption densitometry of fluorescence quenched zones.

A quantitative method using silica gel HPTLC plates with fluorescent indicator, automated sample application, and automated UV absorption densitometry of the fluorescence quenching zones was developed and validated for determination of loperamide hydrochloride in anti-diarrheal medications. Samples of three brands of caplets assayed within 96.0-105% of the 2 mg label value. Repeatability was 3.3%, 1.6%, and 2.8% (RSD) for replicate analyses (n=6) of three tablets. The errors of a blank-spike and standard analyses performed to evaluate accuracy were 2.00% and 2.02%, respectively. The method is suitable for application in a drug manufacturing quality control or regulatory analysis laboratory.

Analytical studies on the charge transfer complexes of loperamide hydrochloride and trimebutine drugs. Spectroscopic and thermal characterization of CT complexes.

Charge transfer complexes of loperamide hydrochloride (LOP.HCl) and trimebutine (TB) drugs as electron donor with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), tetracyanoethylene (TCNE) and 7,7,8,8-tetracyanoquinodimethane (TCNQ) as π-acceptors in acetonitrile were investigated spectrophotometrically to determine the cited drugs in pure and dosage forms. The reaction gives highly coloured complex species which are measured spectrophotometrically at 460, 415 and 842nm in case of LOP.HCl and at 455, 414 and 842nm in case of TB using DDQ, TCNE and TCNQ reagents, respectively. The optimum experimental conditions have been studied carefully and optimized. Beer's law was obeyed over the concentration ranges of 47.70-381.6, 21.50-150.5 and 10.00-100.0µgmL(-1) for LOP.HCl and 37.85-264.9, 38.75-310.0 and 7.75-155.0µgmL(-1) for TB using DDQ, TCNE and TCNQ reagents, respectively. Sandell sensitivity, standard deviation, relative standard deviation, limit of detection and quantification were calculated. The obtained data refer to high accuracy and precision of the proposed method. These results are also confirmed by inter and intra-day precision with percent recovery of 99.18-101.1% and 99.32-101.4% in case of LOP.HCl and 98.00-102.0% and 97.50-101.4% in case of TB using DDQ, TCNE and TCNQ reagents for intra- and inter-day, respectively. These data were compared with those obtained using official methods for the determination of the cited drugs. The stability constants of the CT complexes were determined. The final products of the reaction were isolated and characterized using FT-IR, (1)H NMR, elemental analysis and thermogravimetric analysis (TG). The stoichiometry and apparent formation constant of the complexes formed were determined by applying the conventional spectrophotometric molar ratio method.

Nano-electromembrane extraction.

The present work has for the first time described nano-electromembrane extraction (nano-EME). In nano-EME, five basic drugs substances were extracted as model analytes from 200 µL acidified sample solution, through a supported liquid membrane (SLM) of 2-nitrophenyl octyl ether (NPOE), and into approximately 8 nL phosphate buffer (pH 2.7) as acceptor phase. The driving force for the extraction was an electrical potential sustained over the SLM. The acceptor phase was located inside a fused silica capillary, and this capillary was also used for the final analysis of the acceptor phase by capillary electrophoresis (CE). In that way the sample preparation performed by nano-EME was coupled directly with a CE separation. Separation performance of 42,000-193,000 theoretical plates could easily be obtained by this direct sample preparation and injection technique that both provided enrichment as well as extraction selectivity. Compared with conventional EME, the acceptor phase volume in nano-EME was down-scaled by a factor of more than 1000. This resulted in a very high enrichment capacity. With loperamide as an example, an enrichment factor exceeding 500 was obtained in only 5 min of extraction. This corresponded to 100-times enrichment per minute of nano-EME. Nano-EME was found to be a very soft extraction technique, and about 99.2-99.9% of the analytes remained in the sample volume of 200 µL. The SLM could be reused for more than 200 nano-EME extractions, and memory effects in the membrane were avoided by effective electro-assisted cleaning, where the electrical potential was actively used to clean the membrane.

Implementation of droplet-membrane-droplet liquid-phase microextraction under stagnant conditions for lab-on-a-chip applications.

In the current work, droplet-membrane-droplet liquid-phase microextraction (LPME) under totally stagnant conditions was presented for the first time. Subsequently, implementation of this concept on a microchip was demonstrated as a miniaturized, on-line sample preparation method. The performance level of the lab-on-a-chip system with integrated microextraction, capillary electrophoresis (CE) and laser-induced fluorescence (LIF) detection in a single miniaturized device was preliminarily investigated and characterized. Extractions under stagnant conditions were performed from 3.5 to 15 microL sample droplets, through a supported liquid membrane (SLM) sustained in the pores of a small piece of a flat polypropylene membrane, and into 3.5-15 microL of acceptor droplet. The basic model analytes pethidine, nortriptyline, methadone, haloperidol, and loperamide were extracted from alkaline sample droplets (pH 12), through 1-octanol as SLM, and into acidified acceptor droplets (pH 2) with recoveries ranging between 13 and 66% after 5 min of operation. For the acidic model analytes Bodipy FL C(5) and Oregon Green 488, the pH conditions were reversed, utilizing an acidic sample droplet and an alkaline acceptor droplet, and 1-octanol as SLM. As a result, recoveries for Bodipy FL C(5) and Oregon Green 488 from human urine were 15 and 25%, respectively.

Desorption electrospray ionisation mass spectrometry and tandem mass spectrometry of low molecular weight synthetic polymers.

A range of low molecular weight synthetic polymers has been characterised by means of desorption electrospray ionisation (DESI) combined with both mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Accurate mass experiments were used to aid the structural determination of some of the oligomeric materials. The polymers analysed were poly(ethylene glycol) (PEG), polypropylene glycol (PPG), poly(methyl methacrylate) (PMMA) and poly(alpha-methyl styrene). An application of the technique for characterisation of a polymer used as part of an active ingredient in a pharmaceutical tablet is described. The mass spectra and tandem mass spectra of all of the polymers were obtained in seconds, indicating the sensitivity of the technique.