Combination of electrochemistry with chemometrics to introduce an efficient analytical method for simultaneous quantification of five opium alkaloids in complex matrices.

For the first time, an analytical methodology based on differential pulse voltammetry (DPV) at a glassy carbon electrode (GCE) and integration of three efficient strategies including variable selection based on ant colony optimization (ACO), mathematical pre-processing selection by genetic algorithm (GA), and sample selection (SS) through a distance-based procedure to improve partial least squares-1 (PLS-1, ACO-GA-SS-PLS-1) multivariate calibration (MVC) for the simultaneous determination of five opium alkaloids including morphine (MOP), noscapine (NOP), thebaine (TEB), codeine (COD), and papaverine (PAP) was used and validated. The baselines of the DPV signals were modeled as a smooth curve, using P-splines, a combination of B-splines and a discrete roughness penalty. After subtraction of the baseline we got a signal with a two-component probability density. One component was for the peaks and it was approximated by a uniform distribution on the potential axis. The other component was for the observed noise around the baseline. Some sources of bi-linearity deviation for electrochemical data were discussed and analyzed. The lack of bi-linearity was tackled by potential shift correction using correlation optimized warping (COW) algorithm. The MVC model was developed as a quinternary calibration model in a blank human serum sample (drug-free) provided by a healthy volunteer to regard the presence of a strong matrix effect which may be caused by the possible interferents present in the serum, and it was validated and tested with two independent sets of analytes mixtures in the blank and actual human serum samples, respectively. Fortunately, the proposed methodology was successful in simultaneous determination of MOP, NOP, TEB, COD, and PAP in both blank and actual human serum samples and its results were satisfactory comparable to those obtained by applying the reference method based on high performance liquid chromatography-ultraviolet detection (HPLC-UV).

Rapid LC-MS/MS method for determination of drotaverine in a bioequivalence study.

A liquid chromatography coupled with tandem mass spectrometry method for the quantification of the antispasmodic drug drotaverine in human plasma was developed and validated according to the current bioanalytical guidelines. The internal standard used was imipramine. The separation was performed on a Kinetex C18 50×3mm, 2.6µm column under isocratic conditions using a mobile phase of 65:35 (v/v) formic acid 0.2% (v/v) in water and acetonitrile at 40°C with a flow rate of 0.4ml/min. The detection of drotaverine and the internal standard was performed in multiple reaction monitoring (MRM) mode using an ion trap mass spectrometer with electrospray ionization, operating in positive mode. The human plasma samples (0.24ml) were deproteinized with methanol and aliquots of 4µl from supernatants obtained after centrifugation were directly injected into the chromatographic system. The method shows a good linearity (r(2)>0.997), precision (CV<6.3%) and accuracy (bias<5.4%) over the range of 2.24-448ng/ml drotaverine in plasma. The recovery was between 91 and 98%. The limit of quantification was 2.24ng/ml. The analysis required only a 3.0min run. The developed and validated method for the determination of drotaverine in human plasma was successfully applied in a bioequivalence study, for analyzing approximately 1000 subject's samples.

Highly flexible and rapidly degradable papaverine-loaded electrospun fibrous membranes for preventing vasospasm and repairing vascular tissue.

Vasospasm is a common post-operative complication after vascular anastomosis. Currently, the main treatment is a local injection of antispasmodic drugs. However, this method has a high rate of relapse and is subject to a large degree of individual variation, and repeated injections cause additional pain for patients. In this study, we developed highly flexible and rapidly degradable papaverine-loaded electrospun fibrous membranes to be wrapped around vascular suturing to prevent vasospasm. Poly-l-lactic acid/polyethylene glycol (PLLA/PEG) electrospun fibers containing papaverine maintained a high degree of flexibility and could withstand any folding, and are therefore suitable for wrapping vascular suturing. A rapid release of papaverine, between 2 and 7 days, was achieved by adjusting the proportions of PEG and PLLA. PLLA electrospun fibers containing 40% PEG (PLLA-40%) could control drug release and polymer degradation most effectively during the first 2 weeks post-operation. Testing using an in vivo rabbit model showed that PLLA-40% fibrous membranes produced significant antispasmodic effect without observable inflammation or hyperplasia, and the fibrous membranes were ideally biodegradable, with no impact on regional blood flow, pressure, vessel diameter or surrounding tissue hyperplasia. Therefore, papaverine-loaded electrospun fibrous membranes show the potential to greatly reduce post-operative vasospasm and maintain regular vascular morphology during antispasmodic therapy.

Simultaneous determination of diclofenac potassium and drotaverine hydrochloride in human plasma using reversed-phase high-performance liquid chromatography.

A simple high-performance liquid chromatographic method with ultraviolet detection is proposed for the estimation of diclofenac potassium and drotaverine hydrochloride in human plasma. Liquid-liquid extraction was carried out with a mixture of dichloromethane-isopropyl alcohol (80:20, v/v). Chromatographic separation of the analytes and internal standard was achieved on an analytical 250 × 4.6 mm i.d. reversed-phase Thermo BDS Hypersil C8 (5 µm particle size) column using a mobile phase of acetonitrile-0.02M ammonium acetate buffer (53:47, v/v) at pH 3.5. The run time was less than 15 min. Column eluate was monitored at 230 nm. The linearity over the concentration ranges of 25-1500 ng/mL and 32-960 ng/mL was obtained for diclofenac potassium and drotaverine hydrochloride, respectively. The limit of quantification was 25 and 32 ng/mL for diclofenac potassium and drotaverine hydrochloride, respectively. Recoveries of diclofenac potassium and drotaverine hydrochloride from plasma were 97.45% and 98.27%, respectively.

[Influence of Perftoran nanoemulsion on blood plasma concentrations of lipophilic drugs].

The influence of perfluorocarbon blood substitute Perfloran on the plasma concentrations of bendazole, drotaverine, ketorolac and verapamil upon intravenous introduction after Perfloran infusion (5 ml/kg) has been investigated on rabbits. It has been found that the plasma concentrations of verapamil, drotaverine and bendazole (highly lipophilic drugs with log(P) = 4.5, 4.9 and 3.5, respectively) increased in the presence of Perfloran. The influence of Perfloran on the concentration of weakly lipophilic ketorolac was less significant. Perfloran effectively bound drotaverine, ketorolac and verapamil in vitro, whereas the binding of ketorolac by the emulsion particles was weak. Evidently, the infusion of hydrophobic nanoemulsion Perftoran elevates the sorption capacity of plasma and creates prerequisites for the redistribution drugs and favors increase in their concentrations.

Carbon-11 labeled papaverine as a PET tracer for imaging PDE10A: radiosynthesis, in vitro and in vivo evaluation.

Papaverine, 1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline, a specific inhibitor of phosphodiesterase (PDE) 10A with IC(50) values of 36 nM for PDE10A, 1,300 nM for PDE3A and 320 nM for PDE4D, has served as a useful pharmaceutical tool to study the physiological role of PDE10A. Here, we report the radiosynthesis of [(11)C]papaverine and the in vitro and in vivo evaluation of [(11)C]papaverine as a potential positron emission tomography (PET) radiotracer for imaging PDE10A in the central nervous system (CNS). The radiosynthesis of papaverine with (11)C was achieved by O-methylation of the corresponding des-methyl precursor with [(11)C]methyl iodide. [(11)C]papaverine was obtained with approximately 70% radiochemical yield and a specific activity >10 Ci/mumol. In vitro autoradiography studies of rat and monkey brain sections revealed selective binding of [(11)C]papaverine to PDE10A enriched regions: the striatum of rat brain and the caudate and putamen of rhesus monkey brain. The biodistribution of [(11)C]papaverine in rats at 5 min demonstrated an initially higher accumulation in striatum than in other brain regions, however the washout was rapid. MicroPET imaging studies in rhesus macaques similarly displayed initial specific uptake in the striatum with very rapid clearance of [(11)C]papaverine from brain. Our initial evaluation suggests that despite papaverine's utility for in vitro studies and as a pharmaceutical tool, [(11)C]papaverine is not an ideal radioligand for clinical imaging of PDE10A in the CNS. Analogs of papaverine having a higher potency for inhibiting PDE10A and improved pharmacokinetic properties will be necessary for imaging this enzyme with PET.

Docking simulations and in vitro assay unveil potent inhibitory action of papaverine against protein tyrosine phosphatase 1B.

The structural similarity between papaverine and berberine, a known inhibitor of human protein tyrosine phosphatase 1B (h-PTP 1B), prompted us to investigate the potential of papaverine as h-PTP 1B inhibitor. The investigation included simulated docking experiments to fit papaverine into the binding pocket of h-PTP 1B. Papaverine was found to readily dock within the binding pocket of h-PTP 1B in a low energy orientation via an optimal set of attractive interactions. Experimentally, papaverine illustrated potent in vitro inhibitory effect against recombinant h-PTP 1B (IC(50)=1.20 microM). In vivo, papaverine significantly decreased fasting blood glucose level of Balb/c mice. Our findings should encourage screening of other natural alkaloids for possible anti-h-PTP 1B activities.

[Analysis of 37 drugs in whole blood by HPLC after solid phase extraction].

OBJECTIVE: To develop a specific, sensitive, reproducible SPE-HPLC method for the determination of 37 drugs in whole blood. METHODS: With the doxapram as internal standard, Oasis column was used to extract drugs from whole blood. Two kinds of mobile phases were used in this study. Separations were achieved by a LiChrospher 100 RP-C18 (250 mm x 4.0 mm x 5 microm) column kept at 50 degrees C, the DAD detector was set at 230 nm and 250 nm. RESULTS: The limit of detection were 1-30 ng/mL. The method showed excellent linearity and the linear correlation coefficient was > or =0.997 98. The relative standard deviation for between-day and within-day assay were <10%. CONCLUSION: The method is effective, simple, reliable and has been used in real cases.

The significance of putative urinary markers of illicit heroin use after consumption of poppy seed products.

After consumption of poppy seeds various substances were detected in urine or blood samples using an immunoassay and a sophisticated liquid chromatographic-tandem mass spectrometric procedure. These compounds are widely considered to be putative markers of heroin (HER) abuse whereas acetylcodeine was regarded as a marker for illicit preparations ("street HER"). Besides positive urinary opiate immunoassay results during a 48 hours monitoring period, peak concentrations of morphine (MOR), codeine and their glucuronides appeared 4 to 8 hours after ingestion of poppy seeds, and concentrations of total MOR higher than 10 microg/mL were observed. Also, in serum samples taken up to 6 hours after consumption, MOR glucuronides were found. Free MOR was only detected in traces (1 to 3 ng/mL) within 2 hours of consumption. In addition, 3 of 6 onsite opiate sweat tests revealed positive results 6.5 hours after ingestion. Furthermore, it was demonstrated that neither noscapine (NOS) nor papaverine (PAP) was detectable in urine or blood samples after the consumption of poppy seeds containing up to 94 microg NOS and up to 3.3 mug PAP. NOS and PAP were rapidly metabolized, whereas desmethylpapaverine and, especially, its glucuronide were found in urine samples of poppy seed consumers even 48 hours after consumption. According to these results PAP metabolites should not be regarded as markers of illicit HER abuse. In conclusion, only acetylcodeine can be regarded as a specific marker but has the problem of a short half-life. Therefore, we suggest that NOS and PAP, but not their metabolites, might be used cautiously as additional markers of illicit HER abuse as they have not been detected after oral intake of poppy seeds in normal doses. But it must be kept in mind that in some cases poppy seeds with an unusually high content of these alkaloids could be available, and that these substances are also agents in some pharmaceuticals.

Application of new membrane selective electrodes for the determination of drotaverine hydrochloride in tablets and plasma.

The construction and electrochemical response characteristics of poly vinyl chloride (PVC) membrane sensors for the determination of drotaverine hydrochloride were described. The sensors are based on the use of the ion association complexes of drotaverine cation with sodium phosphotungestate (Dro-PTA) or ammonium reineckate (Dro-R) counter anions as ion exchange sites in the PVC matrix. The performance characteristics of these sensors, which were evaluated according to IUPAC recommendations, reveal a fast, stable and linear response for drotaverine over the concentration range 10(-5) to 10(-2) M with cationic slopes of 49.55 and 51.36 mV per concentration decade. The direct potentiometric determination of drotaverine hydrochloride using the proposed sensors gave average recoveries of 99.95+/-0.71 and 100.04+/-0.60 for Dro-PTA and Dro-R, respectively. The sensors are used for determination of drotaverine hydrochloride in tablets, in its mixture with caffeine and paracetamol and in plasma. Validation of the method shows suitability of the proposed sensors for use in the quality control assessment of drotaverine hydrochloride. The developed method was found to be simple, accurate and precise when compared with a reported HPLC method.

Tissue distribution of moxaverine-hydrochloride in the rabbit eye and plasma.

OBJECTIVE: The aim of this study was to determine the tissue distribution and epithelial penetration of moxaverine-hydrochloride (MOX) in the rabbit eye. METHODS: For systemic application, a radioactively labeled MOX solution was injected into the ear vein of Dutch-belted pigmented male rabbits. For topical dosing, an identical solution was administered. At predetermined time points, rabbits were sacrificed, the eyes dissected, and the amount of MOX in the ocular tissues measured. To examine the MOX permeability across the corneal epithelium, transport studies using rabbit corneal epithelial cell culture were conducted and the respective apparent permeability coefficient in absorptive (a to b) or secretive (b to a) direction was calculated. RESULTS: Topical delivery resulted in high concentrations of MOX in the cornea and conjunctiva, although other tissues of the anterior part yielded lower MOX concentrations. In the tissues of the posterior part, high amounts were detected in the retina. Plasma levels were low. The apparent permeability coefficient across corneal epithelial cell layers was in the range of 10(5) cm/s, exhibiting no apparent directionality. CONCLUSION: A topical dosing of MOX to posterior regions of the eye seems feasible. MOX levels in the posterior part of the eye were remarkably high, without causing stringent plasma levels. The high apparent permeability coefficient of MOX across the corneal epithelial cell layers might be caused by the lipophilic nature of the drug and was in the range of other compounds with comparable physicochemical properties.

[Determination of papaverine hydrochloride in skin and blood and the drug contents in pig skin].

OBJECTIVE: To explore the application of ultraviolet spectrophotometer in determining papaverine hydrochloride content in blood and skin and to determine the intradermal content after local external application of papaverine hydrochloride. METHODS: Phosphate buffer solution (pH = 10, ionic strength = 0.4) was added into the blood or ground skin tissue containing papaverine hydrochloride, and papaverine hydrochloride was extracted with chloroform-hexane mixture (ratio:2:3). After oscillation and centrifugation, partial organic phase was retrieved and dried in 60 degrees C water bath. The dried product was dissolved in 1 mol/L HCl and then underwent filtration through 0.2 micron membrane. The ultraviolet apectrophotometry was applied to determine the content of papaverine hydrochloride in the filtrate. 2% papaverine hydrochloride cream was applied on pig's skin and skin specimen was retrieved at given time to determine the papaverine hydrochloride content in skin tissue with ultraviolet spectrophotometry. RESULTS: The absorbance spectrum of the extract was typical of that of papaverine hydrochloride, with no miscellaneous peak. The extractive rate of papaverine hydrochloride in blood of was more than 92% and that in skin tissue more than 87%. Eight hours after external application of 2% papaverine hydrochloride cream on pig skin, the content of papaverine hydrochloride in skin tissue was more than 90 micrograms/g, significantly higher than the effective concentration of papaverine hydrochloride. CONCLUSIONS: Ultraviolet spectrophotometry is a simple, economical and reliable method for the determination of papaverine hydrochloride content in blood and skin tissue, with good reproducibility and sensibility. External application of papaverine hydrocholine cream can provide efficient permeation and drug concentration in the skin.

Evaluation of several solid phase extraction liquid chromatography/tandem mass spectrometry on-line configurations for high-throughput analysis of acidic and basic drugs in rat plasma.

Several configurations using 6- and 10-port switching valves were studied for high flow, on-line extraction of rat plasma coupled to an electrospray triple quadrupole mass spectrometer. Each plasma sample was diluted 1:1 with an aqueous internal standard solution. The sample was injected into a 2.1 x 20 mm cartridge column packed with 25 microm divinylbenzene/N-vinylpyrrolidone packing using 100% aqueous mobile phase at 4 mL/min. After sample loading and sample cleanup, the analytes were eluted from the extraction column with a 1.0-min gradient at 0.4 mL/min. The samples were either analyzed directly after elution from the extraction column or after additional separation using a short high performance liquid chromatography (HPLC) column. The different configurations were tested using an acidic drug (diflunisal) and a basic drug (clemastine) in rat plasma. On-line analysis was performed by injecting 200 microL of diluted plasma. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. All calibration standards gave relative standard deviations (RSDs) below 5%. The total time per sample was 3 min.

Papaverine topical gel for treatment of erectile dysfunction.

Intracavernous injection of vasoactive substances has been shown to be an effective means of treatment of organic erectile dysfunction. However, up to 50% of men eventually discontinue treatment often because of lack of spontaneity and needle phobia. This study was done as a phase I, placebo controlled, nonblinded investigation of the safety and efficacy of a topical papaverine gel in the treatment of erectile dysfunction. Of 20 men with organic impotence 17 completed the trial and 13 of these patients had spinal cord injuries. After application of a 15% and 20% papaverine base gel to the scrotum, perineum and penis, cavernous artery diameter was significantly increased (36%, p < 0.001) as assessed by color flow Doppler ultrasound. Peak systolic flow velocity increased 26%. Only 3 of 14 patients achieved an increase in cavernous artery diameter of 75% or more and 2 of 14 had a peak systolic flow velocity of 25 cm. per second or more after application of a topical base gel. Similar findings were present when only the patients with spinal cord injury were analyzed. The effect of a papaverine base in producing flow alterations to the penis is dose-dependent. A diminution in blood pressure was present at 15 and 30 minutes after application to the forearm, and the heart rate diminished from 68 to 62 beats per minute after application to the genitalia. No patient was symptomatic. Serum papaverine levels were not elevated over pre-application values. No hepatotoxic effects were demonstrated. Full clinical erections (mean duration 38.7 minutes) were present in 3 patients but were also present with the placebo preparation (mean duration 8.0 minutes). In conclusion, topical papaverine gel appears to be safe and well tolerated after application to the genitalia, and increases blood flow to the penis with a 15% and 20% base preparation. Minimal systemic absorption occurs and, thus, effects are probably from local absorption. Topical therapy appears to augment reflex erections in the spinal cord injury patient and may be especially beneficial in this population. Further investigation is warranted at higher concentrations or in combination with different skin absorption enhancers.

High-performance liquid chromatographic method for the determination of drotaverine in human plasma and urine.

A simple and sensitive HPLC method for the determination of drotaverine in human plasma and urine has been developed. Alkalinized plasma or urine was extracted with organic solvent and the basic components in the organic phase were back-extracted into 0.1 M HCl. An aliquot of the aqueous layer was injected onto the column and the eluent was monitored at 254 nm. Separation was performed on a C18-column with 0.02 M sodium dihydrogen phosphate-methanol (30:70, v/v) containing perchlorate ion at pH 3.2 as mobile phase. Drotaverine was well resolved from the plasma constituents and internal standard. An excellent linearity was observed between peak-height ratios and plasma concentrations and the intra- and inter-assay coefficients of variation were always < 10%. The lowest limit of detection (signal-to-noise ratio 3:1) was 6 ng/ml. The method is suitable for therapeutic monitoring and pharmacokinetic studies of drotaverine in humans as well as in animal models.

Modified high-performance liquid chromatographic method for analysis of drotaverine in human plasma.

An HPLC method is described for the determination of drotaverine in plasma; papaverine is used as the internal standard. The lower limit of quantitation is 50 ng ml-1 with an inter-assay precision (RSD) of below 4%. The method has been validated and successfully used to assay clinical trial samples in healthy volunteers.

Pharmacokinetics and bioavailability of papaverine HCl following intravenous, peroral, rectal, vaginal, topical and buccal administration in beagle dogs.

This in vivo study was designed to obtain bioavailability data and a definite pharmacokinetic profile of papaverine HCl in Beagle dogs following intravenous (IV), peroral (PO), rectal, vaginal, topical, and buccal administration of different papaverine HCl formulations. Blood samples were analyzed by high-performance liquid chromatography. The pharmacokinetic parameters were determined using either a curve fitting program (RESID) or a compartment model independent program (AUC-RPP). The plasma concentration-time profiles show that papaverine HCl pharmacokinetics is best described by an open two-compartment model. The absolute bioavailability of papaverine HCl was determined to be 57.2 per cent, 25.2 per cent, 53.2 per cent, 3.2 per cent and 7.5 per cent, respectively, following P.O., rectal, vaginal, topical and buccal administration.

[An attempt to classify vascular impotence using penile erection curve patterns].

Papaverine hydrochloride was administered to the cavernous body of the penis in a total of 262 patients who were diagnosed as impotent at our Reproduction Center, Toho University, during the 5 years' period from 1984 to 1988. The condition of erection was accurately recorded by the use of both a mercury strain gauge and rubber strain gauge to classify the erection curve, and papaverine hydrochloride outflow into the peripheral blood was concurrently measured in some of the cases in order to assess its relation with the erection curve pattern. As for the concentration of papaverine hydrochloride in the peripheral blood, it was found that the better the state of erection, the smaller the outflow of peripheral blood. An assessment of the relationship of the concentration of papaverine hydrochloride with the classification of erection pattern showed that the amount of outflow was smaller in the N type (normal) pattern and that the amount of outflow was increasingly larger in the following order: A type (arterial), N' type (arterial and venous) and V type (venous). The distribution of the erection curve patterns, in the 262 cases was: N type, 45%; A type, 10%; N' type, 22.5%; and V type, 22.5%. From the above findings, it is presumed that classification of erection curve pattern under papaverine hydrochloride loading is useful for the differentiation of vascular impotence, particularly for the diagnosis of disorders in the outflow system.