苍术中非法添加硫酸阿托品及氢溴酸东莨菪碱的检测方法

目的建立中药苍术中非法添加硫酸阿托品及氢溴酸东莨菪碱的定性定量检测方法。方法采用薄层色谱法定性、高效液相色谱法定量;色谱柱为phenomenex Luna C18柱（200 mm×4.6 mm,5μm）,流动相为0.07 mol/L磷酸钠溶液（含0.017 5 mol/L十二烷基硫酸钠,用磷酸调节pH至6.0-乙腈（70∶30）,流速为1.0 mL/min,柱温为30℃,检测波长为216 nm。结果硫酸阿托品及氢溴酸东莨菪碱进样量分别在1.97～9.85μg和2.03～10.15μg的范围内与峰面积积分值线性关系良好,平均加样回收率分别为99.67%和99.77%,RSD为0.12%和0.19%（n=6）。结论该方法简便、快速、准确,可用于检测苍术中非法添加的硫酸阿托品及氢溴酸东莨菪碱。     

Antagonistic effects of antimuscarinic drugs on alpha 1-adrenoceptors

We previously observed that noradrenaline (NA)-induced contraction of the portal vein of rabbit was relaxed by the antimuscarinic drugs of atropine sulfate, but not scopolamine hydrobromide. In the present study we examined the possible effect of the antimuscarinic drugs of atropine sulfate, scopolamine hydrobromide, p-fluoro-hexa-hydro-sila-difenidol ( p-F-HHSiD, the M(3)-receptor antagonist) and pirenzepine (the M(1)-receptor antagonist) on alpha(1)-adrenoceptor (AR).Atropine and p-F-HHSiD relaxed the alpha(1)-AR agonist methoxamine-induced contraction of the rabbit portal vein in a concentration-dependent manner; however, scopolamine and pirenzepine had no such inhibitory effect. Radioligand binding studies with the alpha(1)-AR ligand 2-[2-(4-hydroxy-3-[(125)I]iodo-phenyl)ethylaminomethyl]-alpha-tetralone ([(125)I]HEAT) in membrane preparations from mouse whole brain showed that atropine (p K(i)=5.33) and p-F-HHSiD (p K(i)=5.88) had higher affinities than scopolamine (p K(i)=3.17) and pirenzepine (p K(i)<2.70). Furthermore, atropine and p-F-HHSiD had higher affinities for all human cloned alpha(1)-ARs than scopolamine and pirenzepine. The results show that the antimuscarinic drugs atropine and p-F-HHSiD have a direct but weak antagonistic activity against alpha(1)-ARs.     

Development and validation of an accurate HPLC method for the quantitative determination of picroside II in tablets

A simple, sensitive and accurate high performance liquid chromatographic method (HPLC) with UV detection was developed and validated to determine picroside II in a new tablet formulation with paeoniflorin as internal standard. Chromatographic separation was achieved on an Agilent XDB C18 column (250 x 4.6 mm I.D., 5 microm) using a mobile phase consisting of acetonitrile-water-acetic acid (18:82:0.4, v/v/v) at a flow rate of 1.0 ml/min. The UV detection wavelength was set at 265 nm. Linear calibration curves were obtained in the concentration range of 0.10-100 microg/ml with the limit of quantification (LOQ) of 0.10 microg/ml. The within- and between-run precisions in terms of % relative standard deviation (RSD) were lower than 5.7% and 6.3%, respectively. The accuracy in terms of % relative error (RE) ranged from -2.3% to 5.0%. This validated method was successfully applied to the determination of the content of picroside II in a new tablet formulation.     

高效液相色谱法测定胃痛宁片中天仙子浸膏含量

目的建立测定胃痛宁片中天仙子浸膏含量的高效液相色谱(HPLC)法。方法色谱柱为SunFire C18柱(250 mm×4.6 mm,5μm),流动相为乙腈(A)-0.1%磷酸溶液(B,含0.02%的三乙胺),梯度洗脱,流速为1.0 m L/min,柱温为35℃,检测波长为210 nm。结果氢溴酸东莨菪碱和硫酸阿托品与相邻杂质峰均可以完全分离;氢溴酸东莨菪碱和硫酸阿托品质量浓度分别在0.002146~0.10729 mg/m L和0.002071~0.10354 mg/m L范围内与峰面积线性关系良好,平均回收率分别为100.04%和98.91%,RSD分别为5.86%和5.54%(n=9)。结论该法可有效测定胃痛宁片中天仙子浸膏的含量,可为完善胃痛宁片质量标准提供参考。     

Uniformly sized molecularly imprinted polymer for atropine and its application to the determination of atropine and scopolamine in pharmaceutical preparations containing Scopolia extract

A uniformly sized molecularly imprinted polymer (MIP) for atropine has been prepared. The MIP was prepared using 2-(trifluoromethyl) acrylic acid and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multi-step swelling and thermal polymerization method. The selectivity factor, which is defined as the ratio of the retention factors (k) on the molecularly imprinted and non-imprinted polymers, k(imprinted)/k(non-imprinted), was 2.2 for atropine on the MIP. The obtained MIP was applied for the determination of tropane alkaloids (atropine and scopolamine) in a commercial gastrointestinal drug by a column-switching HPLC system, consisting of an MIP material as a pre-column, and a conventional cation-exchange analytical column. An interference peak was observed at the retention time of atropine derived from pre-column. However, since the peak area was less than 0.5% the peak area of atropine of a standard solution under the analytical conditions of this study (0.2 microg of atropine was loaded), this interference was negligible in the determination of atropine. On the other hand, no interference peak was observed at the retention time of scopolamine. Calibration curves of atropine and scopolamine showed good linearity in the range of 0.02-0.9 microg/ml (r=0.9999) and 0.003-0.09 microg/ml (r=0.9998), respectively. The mean recoveries of atropine and scopolamine from a placebo pharmaceutical preparation sample were 98.9 and 99.9%, respectively. The intra-day precision (measured by relative standard deviation, R.S.D. (%)) of both ingredients was less than 2.0%. The optimized column-switching system was applied successfully to the determination of atropine and scopolamine in a commercial gastrointestinal drug.     

A biochemical study of cholinergic-dopaminergic interactions in the central nervous system

The effects of cholinergic (Ach) and anti-Ach drugs on striatal dopamine (DA) levels and turnover were studied. Treatment with both atropine sulfate and scopolamine hydrobromide increased striatal DA levels; scopolamine increased striatal DA turnover and decreased both striatal 3,4-dihydroxyphenylacetic acid (DOPAC) levels and DOPAC/homovanillic acid (HVA) relation. Oxotremorine decreased striatal DA levels but did not change striatal DA turnover or DA metabolites. Changes in striatal DA levels or turnover were not observed after atropine methyl nitrate or physostigmine. The data show the Ach-DA interactions in the striatum.     

Modulation of cholinergic activity and the aversive threshold in the rat

The analgesic potency of atropine sulfate (5.0, 10.0, 20.0, 40.0 mg/kg), eserine sulfate (0.5, 1.0, 1.5 mg/kg), pilocarpine nitrate (5.0, 10.0, 20.0 mg/kg), scopolamine methylbromide (0.5, 1.0 mg/kg) and scopolamine hydrobromide (1.0 mg/kg) was measured in the rat using the spatial preference technique. Enhanced cholinergic tone via the administration of eserine or pilocarpine in conjunction with scopolamine methylbromide produced significant increments in the aversive threshold. These increments could not be accounted for solely by changes in motor activity or the debilitating effects of enhanced peripheral cholinergic stimulation. None of the anticholinergics tested affected the aversive threshold. Scopolamine hydrobromide (1.0 mg/kg), however, was able to fully block the increments in the aversive threshold noted after the administration of pilocarpine (10.0 mg/kg). These results were interpreted to suggest that agents which enhance cholinergic tone can produce significant analgesia in the rat. While no firm conclusions can be made without further evidence, especially with regard to the antianalgesic effects of the anticholinergics, it is possible that central cholinergic mechanisms may mediate the aversive qualities of electric shock in the rat.     

Simultaneous determination of loratadine and pseudoephedrine sulfate in pharmaceutical formulation by RP-LC and derivative spectrophotometry

Highly sensitive, simple and accurate reversed phase liquid chromatographic and first derivative spectrophotometric methods for determination of antihistaminic drug loratadine [I] and nasal decongestant drug pseudoephedrine sulfate [II] are described. The HPLC method involves separation of [I] and [II] on micro-BondaPak C18 column using mixture of (methanol:H(2)O:phosphoric acid:ammonium dihydrogen phosphate) (220:300:2:3 g) (V/V/V/W), 60 and 40% acetonitrile as mobile phase flowing at 2 ml/min with ultraviolet detection at 247 nm. The calibration graphs are linear from 5 to 100 microg/ml for [I] and from 120 to 1200 microg/ml for [II] the detection limits are 0.5 microg/ml for [I] and 60 microg/ml for [II]. The spectrophotometric method is based on recording the first derivative spectra for [I] and [II] at 307, 266 nm, respectively, of their solutions in 0.1 M hydrochloric acid using the acid as blank. The calibration graphs are linear in the range of 5-25 microg/ml for [I] and 240-720 microg/ml for [II]; the limits of detection are 0.16 microg/ml for [I] and 10 microg/ml for [II]. The mean percentage recoveries obtained for different synthetic mixtures by using this method are 97.6% with coefficient of variation 1.79 for [I] and 101.6% with coefficient of variation 1.95 for [II]. The two methods have been applied successfully for the determination of [I] in its combination with [II] Clarinase tablets and [I] alone in different pharmaceutical dosage forms.     

Validated enantiospecific LC method for determination of (R)-enantiomer impurity in (S)-efavirenz

A high-performance liquid chromatographic method was developed for separation of the enantiomers of efavirenz. The developed method was applied for the determination of (R)-enantiomer in (S)-efavirenz and satisfactory results were achieved. The base line separation with a resolution of more than 4.0 was achieved on Chiralcel OD (250 mm x 4.6 mm, 10 microm) column containing tris-(3,5-dimethylphenylcarbomate) as stationary phase. The mobile phase consists of n-hexane: isopropyl alcohol (80:20 v/v) with 0.1% (v/v) of formic acid as additive. The flow rate was kept at 1.0 ml/min and the UV detection was monitored at 254 nm. The (R)-enantiomer was found linear over the range of 0.1 microg/ml--6 microg/ml. The limit of detection (LOD) was 0.03 microg/ml and the limit of quantification (LOQ) was 0.1 microg/ml (n=3. The precision of (R)-enantiomer at LOQ level was evaluated through six replicate injections and the RSD of the peak response was achieved as 1.34%. The results demonstrated that the developed LC method was simple, precise, robust and applicable for the purity determination of efavirenz.     

氢溴酸东莨菪碱注射液的紫外分光光度法测定

目的：建立氢溴酸东莨菪碱注射的含量测定方法。方法：测定波长为257nm，溶剂为水。结果：在0．2mg/ml-1.2mg/ml范围内，氢溴酸东莨菪碱的浓度与吸光度间具有良好的线性，平均回收率为99．9％，RSD为0．4％。结论：紫外分光光度法可准确测定氢溴酸东莨菪碱注射液的含量。     

Intraventricular anti-cholinergics do not block cholinergic hippocampal RSA or neocortical desynchronization in the rabbit or rat

Electroencephalographic (EEG) electrodes and ventricular cannulae were implnated in 8 rabbits and 12 rabbits. Two anti-cholinergic agents, atropine sulfate and scopolamine hydrobromide, were given systematically (1–50 mg/kg) and intraventricular (5–800 μg). Systematic but not intraventricular injections blocked sensory stimulation-induced or eserine-induced neocortical desynchronization and hippocampal RSA in rats and rabbits which were immobile and either undrugged or ethanol intoxicated. Systemic injections also blocked hippocampal RSA but not neocortical desynchronization in rats given sensory stimulation under urethane anaesthesia, while intraventricular injections only reduced RSA amplitude. Neither systemic nor intraventricular injections blocked neocortical desynchronization or hippocampal RSA recorded from animals when they walked in a motor driven wheel. These experiments support the hypothesis that there are two types of neocortical desynchronization and hippocampal RSA, one cholinergic and one-cholinergic. They also suggest that atropine and scopolamine pass more readily to the neural system responsible for cholinergic EEG activity from the capillary bed than from the ventricular fluid.     

Development and validation of a liquid chromatographic method for determination of enantiomeric purity of citalopram in bulk drugs and pharmaceuticals

A simple, rapid and robust LC method for enantiospecific separation and determination of citalopram in drugs and pharmaceuticals was developed using UV and polarimetric detectors connected in series. Baseline separation with resolution > or = 3.0 was achieved within 20 min on Chiralcel OD-H (250 mm x 4.6 mm) 5 microm column using a mobile phase containing of n-hexane:2-propanol:triethylamine (TEA) (95:05:0.1 v/v/v) at a flow rate of 1.0 ml/min at 25 degrees C. Effects of 2-propanol, triethylamine and temperature on enantioselectivity and resolution of the enantiomers were evaluated. Clopidogrel hydrogen sulphate was used as an internal standard (IS) for quantitative determinations using UV detector at 240 nm. Polarimetric detector was used for identification of enantiomers. The limits of detection (LOD) and quantification (LOQ) were 0.5 and 1.3 microg/ml respectively for both the enantiomers. The linearity of the method was in the range of 50-600 microg/ml with r2 > 0.9999. The inter- and intra-day assay precision was less than 0.63% (%R.S.D.) and recoveries were in the range 99.38-100.41%. The method was validated and found to be suitable for determination enantiomeric purity of citalopram in bulk drugs and pharmaceutical formulations.     

Effects of selected anticholinergics on acoustic startle response in rats

The present study compared the effects of the anticholinergics aprophen hydrochloride, atropine sulfate, azaprophen hydrochloride, benactyzine hydrochloride, biperiden hydrochloride, diazepam, procyclidine hydrochloride, scopolamine hydrobromide and trihexyphenidyl hydrochloride on acoustic startle response in rats. Peak startle amplitude, latency to peak startle amplitude and prepulse inhibition following 100- and 120-dB tones were recorded 15 min following drug administration in food-restricted rats. Aprophen, atropine, azaprophen, benactyzine, biperiden and scopolamine significantly increased peak startle amplitude and decreased latency to peak startle amplitude following 100-dB pulses. In contrast, only biperiden increased peak startle amplitude following 120-dB pulses, whereas atropine and trihexyphenidyl decreased latency to peak startle amplitude following 120-dB pulses. Benactyzine decreased prepulse inhibition following both 100- and 120-dB pulses, whereas both biperiden and scopolamine decreased prepulse inhibition following 120-dB pulses. Acoustic startle response measures were effective in differentiating the effects of anticholinergic compounds. The comparison of drug effects on the acoustic startle response may be useful in selecting efficacious anticholinergic drug therapies with a minimal range of side-effects. In addition, these data may be useful in down-selecting the number of anticholinergic drugs that need to be tested in comparison studies involving more complex behavioral tests.     

Deficits in passive-avoidance learning following atropine in the developing rat

The maturation of cholinergic inhibitory mechanisms that may be involved in passive-avoidance learning was studied in rats 14, 17, 21, 25, 28, and 34 days of age. Acquisition and extinction of the conditioned response were examined under saline and atropine sulfate (5 mg/kg). Learning was also tested following scopolamine hydrobromide injections (1, 4, 8 mg/kg) in rats 17 days of age and following -methylatropine (5 mg/kg) in 17-and 34-day-old groups. In normal animals the rate of acquisition increased during ontogenesis, with a significant improvement between postnatal days 17 and 21, whereas the rate of extinction did not vary with age. Acquisition was impaired by atropine sulfate at all ages and even totally prevented in younger groups (14 and 17 days of age). It was also completely disrupted by scopolamine in 17-day-old rats. Extinction following acquisition under atropine was more rapid than after normal acquisition. Methyl-atropine was without effect. These results support the hypothesis of central cholinergic mechanisms involved in response suppression, already functioning in the rat 14 days of age and maturing mainly between the 17th and the 21st postnatal days.     

Quantitative determination of insulin entrapment efficiency in triblock copolymeric nanoparticles by high-performance liquid chromatography

A rapid and effective isocratic chromatographic procedure was described in this paper for the determination of insulin entrapment efficiency (EE) in triblock copolymeric nanoparticles using reversed-phase high-performance liquid chromatography (RP-HPLC) with an ultraviolet/visible detector at low flow rate. The method has been developed on a Shimadzu Shim-pack VP-ODS column (150 mm x 4.6 mm, 5 microm, Chiyoda-Ku, Tokyo, Japan) using a mixture of 0.2 M sodium sulfate anhydrous solution adjusted to pH 2.3 with phosphoric acid and acetonitrile (73:27, v/v) as mobile phase at the flow rate of 0.8 ml min(-1) and a 214 nm detection. The method was validated in terms of selectivity, linearity, precision, accuracy, solution stability, limit of detection (LOD) and limit of quantification (LOQ). The calibration curve was linear in the concentration range of 2.0-500.0 microg ml(-1), and the limits of detection and quantitation were 8 and 20 ng, respectively. The mean recovery of insulin from spiked samples, in a concentration range of 8-100 microg ml(-1), was 98.96% (R.S.D.= 2.51%, n = 9). The intra- and inter-assay coefficients of variation were less than 2.24%. The proposed method has the advantages of simple pretreatment, rapid isolation, high specificity and precision, which can be used for direct analysis of insulin in commercially available raw materials, formulations of nanoparticles, and drug release as well as stability studies.     

Sterilized ophthalmic hydrogels with hydroxypropyl methylcellulose: determination and mathematical treatment of flow properties

In this article, the influence of electrolytes on the viscosity of sterilized hydrogels containing 60 g/L of hydroxypropyl methylcellulose (HPMC 4000) was investigated. The electrolytes mostly used in ophthalmics were sodium chloride, pilocarpine hydrochloride, atropine sulphate, and scopolamine hydrobromide. The hydrogels were prepared by using stationary method with a long period of storage in the refrigerator following the steam sterilization. Cone and plate rotational viscometry readings were modeled by using the three-parameter power law, which was validated by regression coefficients in the range of 0.9980-0.9999. In comparison to the hydrogel without additives, the significant decrease in viscosity was observed in the presence of pilocarpine hydrochloride in concentrations of 10 g/L and 30 g/L. On the other hand, the addition of sodium chloride, atropine sulphate, and scopolamine hydrobromide resulted in increasing of viscosity of hydrogels. The differences were nonsignificant when the single-point rheological characteristic, the apparent viscosity eta100, was evaluated, in contrast to using the area under rheogram (AUR) data when the significant effect of sodium chloride could be proved. Therefore, in conclusion, the multipoint rheological AUR data could be recommended. In the investigation of gentle plastic rheograms, nevertheless, the allocation of the lower limit of the integration to yield value is emphasized, which prevents the integration of a negative portion of flow curve and, consequently, the lower values of AUR to be obtained.     

Gradient high-performance liquid chromatography for the simultaneous determination of chlorogenic acid and baicalin in plasma and its application in the study of pharmacokinetics in rats

A novel HPLC-UV method was developed for the simultaneous determination of two major active components in Yinhuang injection, chlorogenic acid and baicalin, in rat plasma. Extracted from the plasma samples with methanol-acetonitrile (3:1, v/v), the two compounds were successfully separated using a C18 column with a gradient elution composed of 15 and 54% methanol-acetonitrile (1:1, v/v) in 0.2% (v/v) phosphoric acid water solution (pH 2.0). The flow-rate was set at 1 ml min(-1) and the eluent was detected at 327 nm for chlorogenic acid, 278 nm for baicalin. Puerarin and rutin were used as the internal standards for chlorogenic acid and baicalin, respectively. The method was linear over the range of 0.388-12.4 microg ml(-1), 0.485-124 microg ml(-1) for chlorogenic acid and baicalin, respectively. The correlation coefficient for each analyte was above 0.998. The intra-day and inter-day precisions were better than 7 and 9%, with the relative error ranging from -9.5 to 7.3% and from -4.2 to 1.8%. The limit of detection (LOD) and the limit of quantification (LOQ) for chlorogenic acid and baicalin in plasma were 0.194, 0.122, 0.388 and 0.485 microg ml(-1), respectively. This assay has been successfully applied in the pharmacokinetic study of chlorogenic acid and baicalin in vivo through intravenous administration of Yinhuang injection to rats.     

Changes in schedule-controlled response and schedule-induced drinking after cholinergic blockers in rats

The effects of the tertiary cholinergic blockers atropine sulfate (AT) and scopolamine hydrobromide (SCOP), and the quaternary cholinergic blockers atropine methylnitrate (AT-Mt) and scopolamine butylbromide (SCOP-Bt), on operant response (lever-pressing) and adjunctive drinking in the rat developed under a fixed interval (FI) 1.5 min food reinforcement situation were investigated. The tertiary compounds, particularly SCOP, increased total responses, and produced a dose-related decrease in the index of curvature (IC) for local rates of response during the FI. The quaternary compounds, however, did not produce changes in the total responses and the IC for local rates of response. AT, AT-Mt and SCOP suppressed adjunctive drinking, while SCOP-Bt did not. AT and SCOP, but not AT-Mt and SCOP-Bt, decreased the IC for local rates of drinking. AT-Mt suppressed drinking at a uniform rate throughout the FI. In addition, several doses of AT and SCOP tended to increase licking counts at the drinking spout per 1 ml water intake. The present results suggest that central cholinergic blockade increases the response and suppress adjunctive drinking, and that the peripheral cholinergic blockade suppresses only adjunctive drinking. Moreover, central cholinergic blockade may produce changes in the patterns of response and drinking during the FI, and in the manner of drinking at the drinking spout.     

A semi-automated solid-phase extraction and radioreceptor assay for the analysis of scopolamine in urine and plasma

In a double-blind placebo-controlled cross-over study, we evaluated the therapeutic efficacy of transdermal scopolamine in ten patients with reversible airways obstruction. They received a patch (Scopoderm^® TTS) behind the ear for three days and samples of blood and urine were taken. A highly sensitive method was developed and validated to measure the low levels of free and total scopolamine in urine and plasma. The procedure consisted of a semi-automated solid-phase extraction followed by the analysis using radioreceptor assays. The mean plasma concentrations, taken every third patch day, of free and total scopolamine were 43.6 pg/ml and 229.0 pg/ml, respectively. In 24-h urine, collected every second patch day, 6.3 μg of free scopolamine and 83.4 μg total scopolamine was excreted. This means that 70% of the delivered dose (120 μg in 24 h) is excreted in urine. For urine samples, the limit of detection (LOD) of the assay is 550 pg/ml and the limit of quantitation (LOQ) is 610 pg/ml. For plasma samples, the LOD is 16 pg/ml and the LOQ is 38 pg/ml.