Drug in Adhesive Patch of Zolmitriptan: Formulation and In vitro /In vivo Correlation

The objective of the present study was to develop transdermal patch for zolmitriptan, determine its in vivo absorption using the rabbit skin. Solvent evaporation technique prepared zolmitriptan patch was settled in two-chamber diffusion cell combined with excised rabbit abdomen skin for permeation study. A sufficient cumulative penetration amount of zolmitriptan (258.5 ± 26.9 μg/cm² in 24 h) was achieved by the formulation of 4% zolmitriptan, 10% Azone, and adhesive of DURO-TAK® 87–4098. Pharmacokinetic parameters were determined via i.v. and transdermal administrations using animal model of rabbit. The results revealed that the absolute bioavailability was about 63%. Zolmitriptan could be detected with drug level of 88 ± 51 ng/mL after transdermal administration of 15 min. The in vivo absorption curve obtained by deconvolution approach using WinNonlin® program was correlated well with the in vitro permeation curve, the correlation coefficient R is 0.84, and the result indicated that in vitro skin permeation experiments were useful to predict the in vivo performance. In addition, little skin irritation was found in the irritation study. As a conclusion, the optimized zolmitriptan transdermal patches could effectively deliver adequate drug into systemic circulation in short time without producing any irritation phenomenon and worth to be developed.KEY WORDS: chemical enhancer, drug-in-adhesive patch, in vitro/in vivo correlation, pharmacokinetic, zolmitriptan     

Simple high-performance liquid chromatographic method for the determination of metformin in human plasma

A simple high-performance liquid chromatographic method using ultraviolet detection was developed for the determination of metformin in human plasma. The method entailed direct injection of the plasma sample after deproteination using perchloric acid. The mobile phase comprised 0.01 M potassium dihydrogen orthophosphate (pH 3.5) and acetonitrile (60:40, v/v). Analyses were run at a flow-rate of 1.0 ml/min with the detector operating at a detection wavelength of 234 nm. The method is specific and sensitive, with a quantification limit of approximately 60 ng/ml and a detection limit of 15 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery value was about 97%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The calibration curve was linear over a concentration range of 62.5–4000 ng/ml.     

Quantitation of trimipramine enantiomers in human serum by enantioselective high-performance liquid chromatography and mixed-mode disc solid-phase extraction

A sensitive and stereospecific method for the quantitation of trimipramine enantiomers in human serum was developed. The assay involves the use of a novel mixed-mode disc solid-phase extraction for serum sample clean-up prior to HPLC analysis and is also free of interference from the enantiomers of desmethyltrimipramine, 2-hydroxytrimipramine, and 2-hydroxydesmethyltrimipramine, the three major metabolites of trimipramine. Chromatographic resolution of trimipramine enantiomers was performed on a reversed-phase cellulose-based chiral column (Chiralcel OD-R) under isocratic conditions using a mobile phase consisting of 0.3 M aqueous sodium perchlorate-acetonitrile (58:42, v/v) at a flow-rate of 0.5 ml/min. Recoveries for R- and S-trimipramine enantiomers were in the range of 93–96% at 25–185 ng/ml levels. Intra-day and inter-day precisions calculated as R.S.D. were in the ranges of 0.30-8.00% and 1.60-10.20% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percent error were in the 0.01–2.10% and 1.00–3.00% ranges for both enantiomers, respectively. Linear calibration curves were in the concentration range 15–250 ng/ml for each enantiomer in serum. The limit of quantification of each enantiomer was 15 ng/ml. The detection limit for each enantiomer in serum using a UV detector set at 210 nm was 10 ng/ml (S/N =2). In addition, separation of the enantiomers of desmethyltrimipramine, 2-hydroxytrimipramine, and 2-hydroxydesmethyltrimipramine were investigated. The desmethyltrimipramine enantiomers could be resolved on the Chiralcel OD-R column under the same chromatographic conditions as the trimipramine enantiomers, but the other two metabolite enantiomers required different mobile phases on the Chiralcel OD-R column to achieve satisfactory resolution with R_s values of 1.00.     

Determination of pyronaridine in blood plasma by high-performance liquid chromatography for application in clinical pharmacological studies

A method is described for the determination of pyronaridine in plasma using high-performance liquid chromatography with fluorescence detection. The method involves liquid-liquid extraction with phosphate buffer (pH 6.0, 0.05 M) and diethyl ether-hexane (70:30%, v/v) and chromatographic separation on a C₁₈ column (Nucleosil, 250 × 4.6 mm I.D., 5 μm particle size) with acetonitrile-0.05 M phosphate buffer pH 6.0 (60:40%, v/v) as the mobile phase (1 ml/min) and detection by fluorescence (λ_ex = 267 nm, λ_em = 443 nm). The detector response is linear up to 1000 ng and the overall recoveries pyronaridine and quinine were 90.0 and 60.3%, respectively. The assay procedure was adequately sensitive to measure 10 ng/ml pyronaridine in plasma samples with acceptable precision (< 15% C.V.). The method was found to be suitable for use in clinical pharmacological studies.     

Determination of buprenorphine by high-performance liquid chromatography with fluorescence detection: application to human and rabbit pharmacokinetic studies

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with fluorescence detection was developed for the determination of buprenorphine in human, rabbit, pig and dog plasma. It is comprised of only a one-step extraction procedure with hexane—isoamyl alcohol at pH 9.25 and reversed-phase chromatography on a μPorasil column. The recoveries of buprenorphine and nalbuphine (internal standard) were greater than 90%. Calibration graphs were linear over the concentration range 3–300 ng/ml with a coefficient of variation, both within-day and between-day, of less than 9% at any level. The limit of detection was 1.0 ng/ml of plasma based on a signal-to-noise ratio of 3. Eight other clinically used narcotics were investigated to check for potential interferences and their analytical conditions. The possible decomposed compounds of buprenorphine were also checked for the specificity of this assay. The method has been succesfully applied to the stability and pharmacokinetic studies of buprenorphine. Buprenorphine in plasma did not decompose significantly at −20°C for four weeks. Pharmacokinetic application in six rabbits and a surgical patient revealed that buprenorphine followed a linear three-compartment model with two distribution phases. The two distribution and elimination half-lives and the clearance of buprenorphine were 1.32, 24.8 and 230 min and 224 ml/min in human plasma, and 0.94, 12.5 and 232 min and 30 ml/min in rabbit plasma.     

Determination of 1-methoxy-2-propanol and its metabolite 1,2-propanediol in rat and mouse plasma by gas chromatography

A method utilizing capillary GC and flame ionization detection was developed for the simultaneous determination of 1-methoxy-2-propanol (propylene glycol monomethyl ether; PGME) and its metabolite 1,2-propanediol (propylene glycol; PG) in rat and mouse plasma. The calibration graphs for rat and mouse plasma were linear with correlation coefficients at>0.997 over the range 2–700 μg/ml. The limit of quantification was ca. 2 μg/ml (2 ng on-column) for both compounds in plasma of each species. The ranges of the precision and accuracy for PGME were 2.8–8.8% and 3.2–13%, respectively, and for PG were 11–26% and 10–25%, respectively. The recovery of PGME from rat and mouse plasma was ca. 73% and for PG it was ca. 65 and 31% from rat and mouse plasma, respectively. The method was used to study the oral absorption and metabolism of PGME in mice. PGME was readily absorbed and metabolized to PG following oral gavage administration at 90 mg/kg. The maximum concentrations of PGME and PG in plasma were attained at 20 and 30 min following dosing, respectively.     

Analysis of Ecteinascidin 743, a new potent marine-derived anticancer drug, in human plasma by high-performance liquid chromatography in combination with solid-phase extraction

A reversed-phase high-performance liquid chromatographic method has been developed and validated for the quantification of the novel anticancer drug Ecteinascidin 743 in human plasma. The sample pretreatment of the plasma samples involved a solid-phase extraction (SPE) on cyano columns. Propyl-p-hydroxybenzoate was added after the sample pretreatment to correct for variability in injection volumes. The separation was performed on a Zorbax SB-C₁₈ column (75×4.6 mm I.D., particle size 3.5 μm) with acetonitrile–25 mM phosphate buffer, pH 5.0 (70:30, v/v) as the mobile phase. The flow-rate was 1.0 ml/min and the eluent was monitored at 210 nm. The accuracies and precisions of the assay fall within ±15% for all quality control samples and within ±20% for the lower limit of quantitation, which was 1.0 ng/ml using 500 μl of plasma. The overall recovery of the sample pretreatment procedure for Ecteinascidin 743 was 87.0±5.9%. The drug was found to be stable in human plasma at −30°C for at least 2 months. At room temperature Ecteinascidin 743 was stable in human plasma for 5 h at most.     

Chromatographic separation of chlorthalidone enantiomers using β-cyclodextrins as chiral additives

Different β-cyclodextrins have been tested as chiral additives in the mobile phase for the chromatographic analysis of chlorthalidone enantiomers in a C₁₈ LiChrospher (125×4 mm I.D.) column. The effect on enantioresolution of different parameters was studied: composition of the mobile phase (percentage of organic solvent, type of buffer and pH), mobile phase flow-rate, and type and concentration of β-cyclodextrin. A 25:75 mixture of methanol and 0.1 M phosphate buffer, pH 4, containing 2% triethylamine (v/v), and 12.5 mM β-cyclodextrin, at a flow-rate of 0.8 ml/min, was found to be the best option for the resolution of chlorthalidone enantiomers. Under such conditions, linear calibration curves were obtained in the 0.5–20-μg/ml interval using UV detection at 230 nm. The limit of detection for both isomers was 50 ng/ml. The utility of the described assay has been tested by analyzing chlorthalidone in different pharmaceutical preparations. Examples of application to biological samples are also given.     

Simple and rapid determination of irinotecan and its metabolite SN-38 in plasma by high-performance liquid-chromatography: application to clinical pharmacokinetic studies

Irinotecan (CPT-11) is an anticancer agent widely employed in the treatment of colorectal carcinoma. A simple, rapid and sensitive high-performance liquid chromatographic method for the simultaneous determination of CPT-11 and its metabolite SN-38 in plasma, and their preliminary clinical pharmacokinetics are described. Both deproteinisation of plasma specimens (100 μl) and addition of the internal standard, camptothecin (CPT), are achieved by incorporating to samples 100 μl of a solution of CPT (1 μg/ml) in acetonitrile–1 mM orthophosphoric acid (90:10); 200 μl of this acidified acetonitrile solution, drug-free, is also added to accomplish complete deproteinisation: this procedure reduces sample preparation time to a minimum. After deproteinisation, samples are treated with potassium dihydrogenphosphate (0.1 M) and injected into a Nucleosil C₁₈ (5 μm, 250×4.0 mm) column. Mobile phase consists of potassium dihydrogenphosphate (0.1 M)–acetonitrile (67:33), at a flow-rate of 1 ml/min. CPT-11, SN-38 and CPT are detected by fluorescence with excitation wavelength set at 228 nm and emission wavelengths of CPT-11, SN-38 and CPT fixed, respectively, at 450, 543 and 433 nm. The limits of quantitation for CPT-11 and SN-38 are 1.0 and 0.5 ng/ml, respectively. This method shows good precision: the within day relative standard deviation (RSD) for CPT-11 (1–10 000 ng/ml) is 5.17% (range 2.15–8.27%) and for SN-38 (0.5–400 ng/ml) is 4.33% (1.32–7.78%); the between-day RSDs for CPT-11 and SN-38, in the previously described ranges, are 6.82% (5.03–10.8%) and 4.94% (2.09–9.30%), respectively. Using this assay, plasma pharmacokinetics of CPT-11, SN-38 and its glucuronidated form, SN-38G, have been determined in one patient receiving 200 mg/m² of CPT-11 as a 90 min intravenous infusion. The peak plasma concentration of CPT-11 at the end of the infusion is 3800 ng/ml. Plasma decay is biphasic with a terminal half-life of 11.6 h. The volume of distribution at steady state (V_ss) is 203 l/m², and the total body clearance (Cl) is 14.8 l/h·m². The maximum concentrations of SN-38 and SN-38G reach 28.9 and 151 ng/ml, respectively.     

Sensitive liquid chromatography assay with ultraviolet detection for a new phosphodiesterase V inhibitor, DA-8159, in human plasma and urine

A sensitive high-performance liquid chromatographic (HPLC) method with ultraviolet absorption detection (292 nm) was developed and validated for the determination of the new phosphodiesterase V inhibitor, DA-8159 (DA), in human plasma and urine. A single step liquid-liquid extraction procedure using ethyl ether was performed to recover DA and the internal standard (sildenafil citrate) from 1.0 ml of biological matrices combined with 200 microl of 0.1M sodium carbonate buffer. A Capcell Pak C18 UG120 column (150 mm x 4.6 mm I.D., 5 microm) was used as a stationary phase and the mobile phase consisted of 30% acetonitrile and 70% 20mM potassium phosphate buffer (pH 4.5) at a flow rate of 1.0 ml/min. The lower limit for quantification was 5 ng/ml for plasma and 10 ng/ml for urine samples. Within- and between-run accuracy and precision were < or =15 and < or =10%, respectively, in both plasma and urine samples. The recovery of DA from human plasma and urine was greater than 70%. Separate stability studies showed that DA is stable under the conditions of analysis. This validated assay was used for the pharmacokinetic analysis of DA during a phase I, rising dose study.     

Sensitive assay for midazolam and its metabolite 1′-hydroxymidazolam in human plasma by capillary high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method is described for the quantification of midazolam and 1′-hydroxymidazolam in human plasma. Sample (1 ml plasma) preparation involved a simple solvent extraction step with a recovery of approximately 90% for both compounds. An aliquot of the dissolved residue was injected onto a 3 μm capillary C₁₈ column (150 mm×0.8 mm I.D.). A gradient elution was used. The initial mobile phase composition (phosphate buffer–acetonitrile, 65:35) was maintained during 16 min and was then changed linearly during a 1-min period to phosphate buffer–acetonitrile, 40:60. The flow-rate of the mobile phase was 16 μl/min and the eluate was monitored by UV detection. The limits of quantification for midazolam and 1′-hydroxymidazolam were 1 ng/ml and 0.5 ng/ml, respectively. The applicability of the method was demonstrated by studying the pharmacokinetics of midazolam, and its major metabolite 1′-hydroxymidazolam, in human volunteers following i.v. bolus administration of a subtherapeutic midazolam dose (40 μg/kg).     

Determination of aspirin and salicylic acid in transdermal perfusates

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of aspirin and salicylic acid in transdermal perfusates. The compounds were separated on a C₈ Nucleosil column (5 μm, 250×4.6 mm) using a mobile phase containing a mixture of water–acetonitrile–orthophosphoric acid (650:350:2, v/v/v) and a flow-rate of 1 ml/min. The transdermal samples were in phosphate-buffered saline (PBS) and could be injected directly onto the HPLC system. The method was reproducible with inter-day R.S.D. values of no greater than 3.46 and 2.60% for aspirin and salicylic acid, respectively. The method was linear over the concentration range 0.2–5.0 μg/ml and had a limit of detection of 0.05 μg/ml for both compounds. For certain samples, it was necessary to ensure that no transmembrane leakage of the aspirin prodrugs had occurred. In these cases, a gradient was introduced by increasing the acetonitrile content of the mobile phase after the salicylic acid had eluted. The method has been applied to the determination of aspirin and salicylic acid in PBS following in vitro application of the compounds to mouse skin samples.     

Rapid high-performance liquid chromatographic method for the separation of hydroxylated testosterone metabolites

A rapid high-performance liquid chromatography (HPLC) method is described for the quantitation of hydroxytestosterone metabolites. The method combines a Hypersil BDS C₁₈ analytical column (10 cm×0.46 cm) and a linear mobile phase (1.25 ml/min) gradient of tetrahydrofuran–acetonitrile–water (10:10:80, v/v) changing to tetrahydrofuran–acetonitrile–water (14:14:72, v/v) over 10 min then remaining isocratic for 3 min. The total run time for the chromatographic separation of eight metabolites of testosterone is 15 min. Detection by UV is linear between 300 ng/ml and 10 μg/ml with a limit of detection on column of 300 ng/ml. A method for the direct HPLC analysis of liver microsomal incubates of [¹⁴C]testosterone is also briefly described and when combined with the HPLC method, offers a distinct advantage over previously reported methods for the rapid screening of testosterone hydroxylase activity in rat and human liver microsomes.     

HPLC method for determination of in vitro delivery through and into porcine skin of adefovir (PMEA)

A simple HPLC/UV method for the determination of the transdermal permeation and dermal penetration of a broad-spectrum antiviral drug adefovir (PMEA) was developed. The separation was achieved on a C18 column with the mobile phase composed of 10 mM KH2PO4 and 2 mM Bu4NHSO4 at pH 6.0 and 7% acetonitrile. The method was validated with respect to selectivity, linearity (0.1-50 microg/ml), precision, accuracy, and stability. Transdermal permeation of 2% PMEA was studied in vitro using the Franz diffusion cell and porcine skin. The flux values were 1.8, 3.0, and 0.6 microg/cm2/h from aqueous donor samples at pH 3.4 and 7.4, and isopropyl myristate, respectively. The respective skin concentrations at 48 h were 294, 263, and 971 microg/g from these vehicles. These results will serve as a lead for further studies on transdermal and topical delivery of antivirals from the group of acyclic nucleoside phosphonates.     

Rapid determination of ranitidine in human plasma by high-performance liquid chromatography without solvent extraction

A simple high-performance liquid chromatographic procedure was developed for the determination of ranitidine in human plasma. The method entailed direct injection of the plasma samples after deproteination using perchloric acid. The chromatographic separation was accomplished with an isocratic elution using mobile phase consisting of 21 mM disodium hydrogen phosphate–triethylamine-acetonitrile (1000:60:150, v/v), pH 3.5. Analyses were run at a flow-rate of 1.3 ml/min using a μbondapak C₁₈ column and ultraviolet detection at a wavelength of 320 nm. The method was specific and sensitive, with a quantification limit of approximately 20 ng/ml and a detection limit of 5 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery was about 96%, while the within- and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The linearity was assessed in the range of 20–1000 ng/ml plasma, with a correlation coefficient of greater than 0.999. This method has been used to analyze several hundred human plasma samples for bioavailability studies.     

Fast determination of ochratoxin A in serum by liquid chromatography: comparison with enzymic spectrofluorimetric method

A rapid high-performance liquid chromatographic method for the determination of low concentrations of ochratoxin A in serum is described. The extraction procedure was simple and short, and liquid chromatographic analysis was carried out isocratically on a reversed-phase C₁₈ column, with methanol—water—acetic acid (30:70:1) as mobile phase and fluorescence detection (excitation at 336 nm, emission at 465 nm). The examined concentration range, 5–50 ng/ml ochratoxin, the recovery method was 87–94%, compared with 62–67% for the enzymic spectrofluorimetric method. The high-performance liquid chromatographic method was faster because the extraction procedure was shorter, and more sensitive so that small sample volumes could be used.     

Simple high-performance liquid chromatographic method for the determination of ranitidine in human plasma

A simple high-performance liquid chromatographic method was developed for the determination of ranitidine in human plasma. Prior to analysis, ranitidine and the internal standard (metoprolol) were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M potassium dihydrogenphosphate–acetonitrile (88:12, v/v) adjusted to pH 6.5. Analysis was run at a flow-rate of 1.3 ml/min and at a detection wavelength of 229 nm. The method is sensitive with a detection limit of 1 ng/ml at a signal-to-noise ratio of 3:1, while the quantification limit was set at 15 ng/ml. The calibration curve was linear over a concentration range of 15–2000 ng/ml. Mean recovery value of the extraction procedure was about 90%, while the within-day and between-day coefficients of variation and percent error values of the assay method were all less than 15%.     

Development of W/O Microemulsion for Transdermal Delivery of Iodide Ions

The objective of this study was to develop a water-in-oil (w/o) microemulsion which can be utilized as a transdermal delivery for iodide ions. Several w/o microemulsion formulations were prepared utilizing Span 20, ethanol, Capryol 90®, and water. The selected formulations had 5%, 10%, 15%, 20%, and a maximum of 23% w/w water content. Potassium iodide (KI) was incorporated in all formulations at 5% w/v. Physicochemical characterizations were conducted to evaluate the structure and stability. These studies included: mean droplet size, pH, viscosity, conductivity, and chemical stability tests. In vitro human skin permeation studies were conducted to evaluate the diffusion of the iodide ion through human skin. The w/o microemulsion formulations were stable and compatible with iodide ions with water content ranging from 5% to 23% w/w. The addition of KI influenced the physicochemical properties of microemulsion as compared to blank microemulsion formulations. In vitro human skin permeation studies indicated that selected formulations improved iodide ion diffusion significantly as compared to control (KI solution; P value < 0.05). Iodide ions were entrapped within the aqueous core of w/o microemulsion. Span 20, ethanol and Capryol 90 protected the iodide ions against oxidation and formed a stable microemulsion. It is worth to note that according to Hofmeister series, iodide ions tend to lower the interfacial tension between water and oil and consequently enhance overall stability. This work illustrates that microemulsion system can be utilized as a vehicle for the transdermal administration of iodide.KEY WORDS: iodide, microemulsion, skin permeation, transdermal     

Rapid high-performance liquid chromatographic determination of docetaxel (Taxotere) in plasma using liquid–liquid extraction

A new rapid and sensitive high-performance liquid chromatographic method for analysis of docetaxel (Taxotere) in human plasma was developed and validated. After adding an internal standard (paclitaxel, Taxol), plasma was extracted following a simple liquid–liquid extraction with diethyl ether. Extraction efficiency averaged 95% for docetaxel. Separation was performed using a Nucleosil (C₁₈) 5 μm column, monitored at 227 nm. The isocratic mobile phase consisted of acetonitrile–acetate buffer, pH 5–tetrahydrofuran (45:50:5, v/v) pumped at a flow-rate of 1.8 ml/min. The limit of quantification for docetaxel in plasma was 12.5 ng/ml. Retention times for docetaxel and paclitaxel were 7.7 and 9 min, respectively. Standard curves were linear over a range of 25–1000 ng/ml. This new method is rapid since it does not require time-consuming extraction procedures, or complex chromatographic conditions. This rapidity, along with the lack of chromatographic interferences with various other drugs likely to be administered to the cancer patients (pain killers, corticoids, antiemetics drugs) make this method suitable for daily routine analysis of Taxotere, a major anticancer drug extensively used in clinical oncology.     

Determination of clozapine and its major metabolites in human plasma and red blood cells by high-performance liquid chromatography with ultraviolet absorbance detection

An isocratic high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantification of clozapine (8-chloro-11-(4′-methyl)piperazino-5H-dibenzo[b,e]-1,4-diazepine) and its two major metabolites in plasma and red blood cells (RBCs). The method involves sample clean-up by liquid-liquid extraction with ethyl acetate. The organic phase was back-extracted with 0.1 M hydrochloric acid. Loxapine served as the internal standard. The analytes were separated by HPLC on a Kromasil Ultrabas C₁₈ analytical column (5 μm particle size; 250×4.6 mm I.D.) using acetonitrile-phosphate buffer pH 7.0 (48:52, v/v) as eluent and were measured by UV absorbance detection at 254 nm. The limits of quantification were 20 ng/ml for clozapine and N-desmethylclozapine and 30 ng/ml for clozapine N-oxide. Recovery from plasma or RBCs proved to be higher than 62%. Precision, expressed as % C.V., was in the range 0.6–15%. Accuracy ranged from 96 to 105%. The method's ability to quantify clozapine and two major metabolites simultaneously with precision, accuracy and sensitivity makes it useful in therapeutic drug monitoring.