X射线荧光光谱法在药品元素杂质分析中的应用

X射线荧光光谱（X-ray Fluorescence Spectrometry,XRF）是基于测量由初级X射线激发的原子内层壳电子产生的特征X射线光量子的一种仪器分析方法。本文介绍了XRF的分类、特点和各国药典标准,综述了国内外XRF在药品研发以及原料药、药用辅料和制剂元素杂质分析中的应用进展,以期为药品的元素杂质控制研究提供参考。     

Potential of synchrotron X-ray powder diffractometry for detection and quantification of small amounts of crystalline drug substances in pharmaceutical tablets

The purpose of this study was to develop a sensitive system for detection of crystalline drug substances in intact pharmaceutical tablets by X-ray powder diffractometry (XRPD), using synchrotron X-rays. Fenoprofen calcium dihydrate was used as a model compound. The wavelength and path length of X-rays from synchrotron radiation were optimized in order to maximize the potential of the synchrotron radiation. The optimum wavelength and path length for the measurement of fenoprofen calcium dihydrate were found to be 0.69817 Å and 6.0 mm, respectively, based on theoretical calculations. Under the optimized conditions, a limit of quantification of 0.05% (RSD = 9.4%, n = 3) and a limit of detection of 0.02% (RSD = 17.3%, n = 3), results which are approximately 10² times as sensitive as those obtained using conventional XRPD instruments, were achieved. The technique was also applied to fenoprofen calcium dihydrate detection in intact film-coated tablets, which contained Ti in the coating film, and a limit of detection of 0.02% was again attained.     

Noninvasive in Vivo Percutaneous Absorption Measurements Using X-Ray Fluorescence

X-ray fluorescence (XRF) has been used to determine in vivo the percutaneous absorption of 5-iodouracil (5IU) in dimethyl sulfoxide (DMSO) on female Sprague-Dawley rats. An average absorption rate constant of 122 ± 34 µg/cm²-hr was obtained from the XRF measurements on four rats. A comparative study was performed with radiolabeled (¹²⁵I) 5IU in which the absorption rate constant was determined to be 126 ± 20 µg/cm²-hr. The XRF system described provides a simple, noninvasive means of measuring the percutaneous absorption rate of select compounds by the surface disappearance method.     

Monitoring the retention of a protein antigen in complete Freund's adjuvant, alum, and pluronic F-127 gel formulations by X-ray fluorescence.

Adjuvants function by protecting antigens from rapid degradation or dispersal. The effectiveness of experimental adjuvants can be assessed by measuring antibody titers to the antigen of interest or, less frequently, by evaluating the retention and distribution of antigen at the application site. In this study, we used X-ray fluorescence (XRF) to monitor the release of an iodinated protein (I-bovine serum albumin) from several adjuvant formulations after its subcutaneous injection in rats. The interaction of the tagged antigen with an external Am-241 source leads to the emission of iodine X-rays from the application site; the number of these X-rays is proportional to the concentration of the protein remaining at the injection site. The disappearance of the iodine X-rays, and hence the antigen, from the injection site followed first-order kinetics for all adjuvant formulations tested; mean half-life values were as follows: in 50% Freund's adjuvant, 17.1 +/- 1.1 h; in 4-hour-old 25% Alum, 11.78 +/- 0.08 h; in 4-h-old 50% Alum, 13.2 +/- 2 h; in 3-day-old 50% Alum, 15.8 +/- 1.5 h; and in 240 mg/mL Pluronic F-127, 7.9 +/- 0.7 h. We conclude that XRF is an easy, reliable, noninvasive method to monitor the retention of antigens in these adjuvant solutions.     

Monitoring the Retention of a Protein Antigen in Complete Freund's Adjuvant,Alum, and Pluronic F-127 Gel Formulations by X-ray Fluorescence

Adjuvants function by protecting antigens from rapid degradation or dispersal. The effectiveness of experimental adjuvants can be assessed by measuring antibody titers to the antigen of interest or, less frequently, by evaluating the retention and distribution of antigen at the application site. In this study, we used X-ray fluorescence (XRF) to monitor the release of an iodinated protein (I-bovine serum albumin) from several adjuvant formulations after its subcutaneous injection in rats. The interaction of the tagged antigen with an external Am-241 source leads to the emission of iodine X-rays from the application site; the number of these X-rays is proportional to the concentration of the protein remaining at the injection site. The disappearance of the iodine X-rays, and hence the antigen, from the injection site followed first-order kinetics for all adjuvant formulations tested; mean half-life values were as follows: in 50% Freund's adjuvant, 17.1 ± 1.1 h; in 4-hour-old 25% Alum, 11.78 ± 0.08 h; in 4-h-old 50% Alum, 13.2 ± 2 h; in 3-day-old 50% Alum, 15.8 ± 1.5 h; and in 240 mg/mL Pluronic F-127, 7.9 ± 0.7 h. We conclude that XRF is an easy, reliable, noninvasive method to monitor the retention of antigens in these adjuvant solutions.     

Dose Calculations for [131I] Meta-Iodobenzylguanidine-Induced Bystander Effects

Targeted radiotherapy is a potentially useful treatment for some cancers and may be potentiated by bystander effects. However, without estimation of absorbed dose, it is difficult to compare the effects with conventional external radiation treatment. Methods: Using the Vynckier – Wambersie dose point kernel, a model for dose rate evaluation was created allowing for calculation of absorbed dose values to two cell lines transfected with the noradrenaline transporter (NAT) gene and treated with [¹³¹I]MIBG. Results: The mean doses required to decrease surviving fractions of UVW/NAT and EJ138/NAT cells, which received medium from [¹³¹I]MIBG-treated cells, to 25 – 30% were 1.6 and 1.7 Gy respectively. The maximum mean dose rates achieved during [¹³¹I]MIBG treatment were 0.09 – 0.75 Gy/h for UVW/NAT and 0.07 – 0.78 Gy/h for EJ138/NAT. These were significantly lower than the external beam gamma radiation dose rate of 15 Gy/h. In the case of control lines which were incapable of [¹³¹I]MIBG uptake the mean absorbed doses following radiopharmaceutical were 0.03 – 0.23 Gy for UVW and 0.03 – 0.32 Gy for EJ138. Conclusion: [¹³¹I]MIBG treatment for ICCM production elicited a bystander dose-response profile similar to that generated by external beam gamma irradiation but with significantly greater cell death.     

Biological fate of 32P malathion in Gallus domesticus (desi poultry birds)

During this study, a minor surgical technique was developed for the separation of urine and faeces in birds and fate of ³²P malathion was studied, following a single oral dose of 394 mg/kg. The birds showed characteristic signs and symptoms of organophosphorus poisoning and the results suggested that the compound is rapidly absorbed from the gastro-intestinal tract, significant quantities being detected in plasma after 0.5 h of ingestion. Highest concentration of ³²P was present during 6 to 8 h of administration. At 6 h, ³²P was highest in liver followed by other organs. With the lapse of time the concentration of ³²P in various organs decreased and at 48 h, it was not detected except in liver, kidney, lung and spleen when only traces were observed. The cumulative urinary and faecal excretion study revealed that within 24 h 90% is rapidly excreted mainly via the urine and only small amounts in the faeces. Metabolism studies showed that the compound is quickly metabolised. Because of the rapid turnover of the compound, this study indicated that the accumulation of this compound is unlikely in the body system.     

Absorption and Elimination Kinetics of 32P Malathion in the Hen

Abstract A method for the separation of urine and faeces in the hen was developed and the biological fate of ³²P malathion, following a single oral dose of 262.40 mg/kg body weight was studied. The results suggested that the compound was rapidly absorbed from the gastrointestinal tract; significant quantities being detected in the plasma and whole blood 1/2 hr after ingestion. Total ³²P was eliminated in the urine by apparent first order kinetics with an average half-life of 5.7 hrs. The cumulative urinary and faecal data revealed that 93 % of the ³²P is excreted via the urine within 48 hrs, thus indicating that the compound is almost completely absorbed. It is therefore concluded that accumulation in the system is unlikely.     

Nitrilotriacetate (NTA): human metabolism and its importance in the total safety evaluation program

Ten milligrams [¹⁴C]NTA in gelatin capsules was given (po) to 8 male subjects. Blood samples were taken, and samples of urine and feces were collected. In addition, expired CO₂ was collected in 4 of the subjects. NTA is poorly absorbed since approximately 12% of the dose appeared in the urine. The compound is rapidly excreted in the urine since 87% of the absorbed dose was excreted within the first 24 hr post dosing. The blood concentration peak occurred 1–2 hr after dosing. Reverse isotope dilution and thin-layer chromatography showed the urinary radioactivity to be unchanged NTA, and therefore no biotransformation had occurred. These results closely resemble the rat and dog metabolic data except that rats and dogs absorb 4 times more NTA than humans. The metabolic similarities among rats, dogs and humans lends some confidence to the extrapolation of the results derived from rat and dog toxicity tests for the purpose of estimating human safety.     

Effects of radioactive contamination on Scots pines in the remote period after the Chernobyl accident

A 6 year study of Scots pine populations inhabiting sites in the Bryansk region of Russia radioactively contaminated as a result of the Chernobyl accident is presented. In six study sites, ¹³⁷Cs activity concentrations and heavy metal content in soils, as well as ¹³⁷Cs, ⁹⁰Sr and heavy metal concentrations in cones were measured. Doses absorbed in reproduction organs of pine trees were calculated using a dosimetric model. The maximum annual dose absorbed at the most contaminated site was about 130 mGy. Occurrence of aberrant cells scored in the root meristem of germinated seeds collected from pine trees growing on radioactively contaminated territories for over 20 years significantly exceeded the reference levels during all 6 years of the study. The data suggest that cytogenetic effects occur in Scots pine populations due to the radioactive contamination. However, no consistent differences in reproductive ability were detected between the impacted and reference populations as measured by the frequency of abortive seeds. Even though the Scots pine populations have occupied radioactively contaminated territories for two decades, there were no clear indications of adaptation to the radiation, when measured by the number of aberrant cells in root meristems of seeds exposed to an additional acute dose of radiation.     

Metabolism and disposition of luminol in the rat

1. The metabolism and disposition of Luminol (LMN, 3-aminophthalhydrazide), a widely used forensic and laboratory reagent that chemiluminesses upon oxidation, was determined as part of its overall toxicological characterization. 2. Radiolabelled LMN was well absorbed, metabolized and excreted following p.o. administration of a range of doses. About 90% of the total dose was recovered within 24 h of administration in urine in the form of two metabolites identified as LMN N⁸-glucuronide and LMN N⁸-sulphamic acid. 3-Aminophthalic acid, the oxidative product of LMN in the light-emitting reaction, was apparently not formed in vivo. 3. Metabolism and disposition of an i.v. administered dose was similar to that following gavage. Little or no LMN-derived radioactivity was present in tissue within 12 h post-dosing. Excretion of radioactivity in bile following i.v. injection was minimal (~8% of the total dose in 6 h) and consisted of the same urinary-excreted glucuronide and sulphate conjugates. 4. LMN was not absorbed dermally in rat, potentially a major route of exposure to human. If the fate of LMN is similar between species, this compound should have little potential for either dermal absorption, bioaccumulation in tissues following other routes of exposure or chronic toxicity in humans.     

X-ray fluorescence elemental mapping and microscopy to follow hepatic disposition of a Gd-based magnetic resonance imaging contrast agent

1. Spatially resolved X-ray fluorescence (XRF) spectroscopy with synchrotron radiation is a technique that allows imaging and quantification of chemical elements in biological specimens with high sensitivity. In the present study, we applied XRF techniques at a macro and micro level to carry out drug distribution studies on ex vivo models to confirm the hepatobiliary disposition of the Gd-based magnetic resonance imaging contrast agent B22956/1. 2. Gd presence was selectively quantified allowing the determination of the time dependent disappearance of the drug from blood and its hepatic accumulation in mice after administration. Elemental mapping highlighted the drug distribution differences between healthy and diseased livers. XRF microanalyses showed that in CCl(4) -induced hepatitis, B22956/1 has greatly reduced hepatic accumulation, shown as a 20-fold reduction of Gd presence. Furthermore, a significant increase of Fe presence was found in steatotic compared with healthy livers, in line with the disease features. 3. The present results show that XRF might be useful in preclinical pharmacological studies with drugs containing exogenous elements. Furthermore, quantitative and high-sensitivity elemental mapping allows simultaneous detection of chemical variation, showing pathological conditions. This approach was useful in suggesting reduced B22956/1 accumulation in steatotic livers, thus opening possible new diagnostic perspectives for this drug.     

Absorption,Excretion and Urinary Metabolic Pattern of 3H-Albuterol Aerosol in Man

1. After inhalation of doses of ³H-albuterol (84 and 220 μg) by humans, plasma radioactivity reached a max. after 2 and 4?h, respectively.

2. About 72% of the inhaled dose was excreted within 24?h in urine and a similar urinary excretion pattern was obtained with both dosages. From the excretion pattern of the unchanged drug in urine a half-life of 3.8?h and an elimination constant of 0.18 h^?1 for albuterol were calc.

3. Approx. 28% dose was excreted as unchanged albuterol, 40% as a major urinary metabolite and 4% as a minor metabolite. The major metabolite could be converted to an albuterol-like compound by acid hydrolysis but not enzymically (Glusulase).

4. These results show that albuterol as an aerosol is readily absorbed, and is excreted mainly in the urine as the free drug and at least two metabolites.     

Pharmacokinetic profile of lesogaberan (AZD3355) in healthy subjects: a novel GABA(B)-receptor agonist reflux inhibitor

Objective: The aim of this study was to evaluate the pharmacokinetic profile of lesogaberan in healthy subjects after single oral and intravenous administration of ¹⁴C-labeled lesogaberan and non-¹⁴C-labeled lesogaberan.Study Design: This was an open-label, single-center, randomized, two-way crossover, phase I study.Participants: Ten healthy male subjects took part in the study.Intervention: Volunteers were randomized to receive a single dose of either orally dosed (100 mg) or intravenously infused (20 mg) non-¹⁴C-labeled lesogaberan, and then orally (100 mg) or intravenously (20 mg) administered ¹⁴C-labeled lesogaberan in a crossover design. Treatment periods were separated by a washout period of at least 7 days.Main Outcome Measures Analyses of the rate and route of excretion, dose recovery, area under the plasma concentration versus time curve (AUC), AUC to the last quantifiable concentration, maximal plasma concentration (C_max), time to C_max, the apparent elimination half-life, bioavailability, total clearance, renal clearance, fraction of the bioavailable dose excreted unchanged in the urine, cumulative amount of drug excreted unchanged in urine, and the apparent volume of distribution at steady state of lesogaberan.Results: Lesogaberan was rapidly and extensively absorbed from the gastrointestinal tract and C_max was achieved within 1–2 hours of oral dosing. The terminal half-life of lesogaberan was between 11 and 13 hours. Renal clearance accounted for approximately 22% of total body clearance. Based on the recovery of administered radioactivity, approximately 84% of the dose was excreted into the urine either as the parent compound or as water-soluble metabolite(s). There were no safety concerns raised during the study.Conclusion: Orally administered lesogaberan is rapidly absorbed with high bioavailability and the majority of the dose is excreted by the kidneys either as the parent compound or as metabolites. The major elimination pathway for lesogaberan in man is metabolism.     

Metabolism of 1,4-Dihydro-6-trifluoromethylquinoxaline-2,3-dione (Lilly 72525) in Rats and Cats

1. The metabolism of 1,4-dihydro-6-trifluoromethylquinoxaline-2,3-dione (Lilly 72525), a sedative hypnotic drug, was studied in rat and cat.

2. Plasma concentrations of Lilly 72525 were measured fluorometrically after oral and intravenous doses of the compound in rats. A comparison of the area under the two curves suggested that 84% of the oral dose was absorbed.

3. Studies with ¹⁴C-labelled material in both species confirmed that the drug was well absorbed after oral administration and revealed that the dione was mainly eliminated unchanged in the urine. Bile duct cannulation experiments suggested that biliary excretion accounted for most or all of the drug present in faeces of rats.

4. Metabolites isolated from urinary extracts by t.l.c. were identified by g.l.c.-mass spectrometry. The only metabolite detected in rat urine or bile extracts was a ring-hydroxylated compound. This metabolite plus two N-hydroxylated metabolites were identified in extracts of cat urine.     

The Fate of 1,3,5-Tri(3,5-Di-Tert-Butyl-4-Hydroxybenzyl)-2,4,6-Trimethylbenzene (Ionox 330) in Rats Fed the Compound over a Prolonged Period

1. The ability of rats to degrade [¹⁴C]Ionox 330 in the alimentary canal was not altered by prior feeding of the compound.

2. [¹⁴C]Ionox 330 was not absorbed from the alimentary canal of pretreated animals.

3. A small quantity of Ionox 330 was oxidized within the alimentary canal followed by a limited absorption of the products.

4. The oxidation of Ionox 330 within the gut and the absorption of products were independent of dose level (20 and 400?mg/kg body weight).

5. It is unlikely that an enzymic mechanism is involved in the initial stage of oxidation of Ionox 330 in vivo     

Antidepressant and antinociceptive activities in animal models and in vitro assessment of the anti-thyroid activity of bis(DL-pyroglutamate)magnesium complex

BackgroundMagnesium is an essential element related with biochemistry of the brain and different types of depression have been associated with its deficiency.MethodsThe structure of a novel magnesium bis(DL-pyroglutamate) (Mg(DL-pGlu)₂) was elucidated by X-ray crystallography. Wistar rats were used in the in vivo experiments. The antidepressant-like effect was assessed by the forced swim test (FST) and the antinociceptive activity was evaluated using hot plate test. In both, non-specific effects were evaluated by the open field test. Anti-thyroid activity was examined using Lang’s method. Albumin binding behavior was evaluated by 3D fluorescence spectroscopy.ResultsFor the Mg(DL-pGlu)₂ complex (30 mg/kg), the FST test on Wistar rats revealed a decrease of 22% in the immobility time and an increment of 106% in the swimming time. The compound alters neither the locomotor activity nor the body weight after chronic administration. At the same dose, it showed antinociceptive activity, increasing the response latency. It blocks iodination reactions generating a charge transfer complex with iodine hence indicating anti-thyroid activity (K_c = 45366.5 ± 29 M^?1). Albumin 3D fluorescence spectroscopy experiments showed intensity increase of peak A and decrease of peak B.ConclusionsThe results showed that the new compound produced a lowering of the immobility time and an increment of the swimming ability of the rats. The compound is able to increase the response latency in 70.0%, to capture iodine (anti-thyroid activity) and to interact with albumin through covalent type of interaction of the free NH groups.     

Release Behaviour of Single Pellets and Internal Fine 3D Structural Features Co-define the In Vitro Drug Release Profile

Multi-pellet formulations are advantageous for the controlled release of drugs over single-unit dosage forms. To understand the diffusion controlled drug release mechanism, the pellet structure and drug release from a single pellet (not at dose level) were studied using synchrotron radiation X-ray computed microtomography (SR-μCT) and a sensitive LC/MS/MS method. The purpose of this article is to introduce a powerful, non-invasive and quantitative technique for studying individual pellet microstructures and to investigate the relationship between the microstructure and drug release from single pellets. The data from the single pellet dissolution measurements demonstrated that the release profile of capsules containing approximately 1,000 pellets per unit dose was the summation of the release profiles of the individual pellets. The release profiles of single tamsulosin hydrochloride (TSH) pellets formed three groups when a cluster analysis was performed, and the dissolution rate of the individual pellets correlated well with the combined effects of the drug loading, volume and surface area of the pellets (R² = 0.9429). In addition, the void microstructures within the pellet were critical during drug release. Therefore, SR-μCT is a powerful tool for quantitatively elucidating the three-dimensional microstructure of the individual pellets; because the microstructure controls drug release, it is an important parameter in the quality control of multi-pellet formulations.KEY WORDS: microstructure, release kinetics, single pellet, synchrotron radiation X-ray computed microtomography     

Absorption of [14C]-Tetrabromodiphenyl Ether (TeBDE) Through Human and Rat Skin In Vitro

The skin is the largest organ in the human body and has the potential to come into contact with a variety of xenobiotics both intentionally (e.g., drugs and cosmetics) or accidentally (e.g., agrochemicals and industrial chemicals). These chemicals may then cross the skin barrier (the stratum corneum) and enter into the systemic circulation where they may produce a desired or an undesired effect, or even no systemic effect at all. Tetrabromodiphenyl ether (TeBDE) is one congener in a mixture of polybrominated diphenyl ethers that makes up a flame-retardant commercial product called pentabromodiphenyl ether (PeBDE). TeBDE was used as a surrogate to assess the potential dermal absorption of this product. The physicochemical properties, including lipophilicity, of TeBDE and PeBDE are similar. Operator exposure of PeBDE product to human skin is possible during production and use. However, during these activities, operators wear protective clothing to protect from or minimize exposure. This study was designed to assess the rate and extent of absorption of [¹⁴C]-tetrabromodiphenyl ether ([¹⁴C]-TeBDE) through human and rat skin in vitro. [¹⁴C]-TeBDE was applied to human and rat split thickness skin membranes in vitro in a single test preparation: [¹⁴C]-TeBDE in acetone (ca. 20%, w/v). Dermal delivery and absorbed dose of TeBDE applied to human skin was 3.13% (313 μg equiv/cm²) and 1.94% (194 μg equiv/cm²) of the applied dose, respectively. Dermal delivery and absorbed dose of TeBDE applied to rat skin was 17.94% (1804 μg equiv/cm²) and 14.81% (1489 μg equiv/cm²) of the applied dose, respectively. These results confirm that the risk of systemic exposure due to external dermal exposure of the PeBDE product is low in the human. Consequently, based on the toxicological profile of these materials, the potential for undesirable effects is also quite low. The results also confirm that the rat is a conservative model overpredicting human absorption about eight fold.     

Pharmacokinetics of pantoprazole following single intravenous and oral administration to healthy male subjects

Summary The plasma pharmacokinetics of pantoprazole have been investigated following single intravenous infusion and single oral administration at a dose of 40 mg to 12 healthy male subjects in a randomised cross-over study. Both treatments were generally well tolerated and no relevant compound-related adverse events were noted. The plasma pharmacokinetics of pantoprazole following intravenous infusion in this group of subjects were characterised by a total plasma clearance of 0.13 l·h⁻¹·kg⁻¹ and apparent terminal elimination half-life 1.9 h. The apparent volume of distribution estimated at steady state (0.171·kg⁻¹) was compatible with the localization of a major fraction of the compound in extracellular water. Following oral administration as an enteric-coated tablet formulation, a variable onset of absorption was followed by rapid attainment of maximum plasma concentrations of pantoprazole. Pantoprazole was well absorbed following oral administration; the absolute systemic bioavailability of the compound was estimated as 77% (95% CI, 67 to 89%).