A validated chiral HPLC method for the enantiomeric separation of tolterodine tartarate

An isocratic chiral HPLC method was developed for the separation of tolterodine tartarate enantiomers. The mobile phase consists of n-hexane and isopropyl alcohol in the ratio of 980:20 (v/v) with 1 ml diethylamine and 0.6 ml trifluoroacetic acid. Chiralcel OD-H (250 mm × 4.6 mm) column was used at constant room temperature. Flow rate was kept at 0.5 ml/min. This method is capable of detecting the S-isomer up to 0.1 μg/ml. The method was validated in terms of linearity, precision, limit of detection (LOD) and limit of quantification (LOQ).     

Spectrophotometric determination of methylene blue in biological fluids after ion-pair extraction and evidence of its adsorption on plastic polymers

Methylene blue has gained a renewed interest in medicine. It is now used in combination with visible light as a photoinactivating agent of membrane viruses such as HIV and HBV in single-donor fresh frozen plasma. MB has also been proposed for the prophylaxis and reversal of ifosfamide induced encephalopathy and in septic shock. In spite of its wide use, its pharmacokinetics in humans are little known and an improved methodology for its quantitation in biological fluids was therefore required. We report an improved spectrophotometric determination of methylene blue in whole blood, plasma and urine. Liquid-liquid ion-pair extraction of MB from the biological matrix is achieved by addition of 500 μl (to 1.2 ml whole-blood aliquot) or 125 μl (to 1 ml plasma or 250 μl urine) of 5% sodium hexanesulfonate solution and subsequent extraction with 2.0 ml 1,2-dichloroethane. After centrifugation, the organic layer is assayed at 657 nm. In urine samples, the reduced form leucomethylene blue is also assayed after oxidation to methylene blue (addition of 38 μl HCl 0.5M to 250 μl urine sample and heating on a water bath at 70°C for 1 min) prior to the ion-pairing extraction. Methylene blue can be accurately determined in spiked whole blood and plasma over the working range 0.1–9.1 μg/ml. At higher concentrations, likely to occur in urine (up to 55 μg/ml), the measurements deviate more than 10% from the nominal values. Analysis of spiked samples shows that methylene blue is adsorbed to some extent to polypropylene and polystyrene plastic tubes. This phenomenon is influenced by the biological matrix and is more marked with low volumes of concentrated (>3 μg/ml) samples. At low concentrations, the adsorption is likely to occur but is more difficult to quantitate due to assay variability. This phenomenon should be kept in mind since it could potentially affect the biological activity of methylene blue.     

Quantitation of bisphenol A and bisphenol A glucuronide in biological samples by high performance liquid chromatography-tandem mass spectrometry.

Bisphenol A (BPA) is a weak estrogen. Pharmacokinetic studies of BPA have demonstrated a rapid and extensive metabolism of BPA to the nonestrogenic BPA-monoglucuronide (BPA-gluc). Some investigators have reported that BPA was found at parts per billion concentrations in the tissues or urine of humans without known exposure to BPA. This work developed a rapid and sensitive method for the determination of BPA and BPA-gluc in plasma and urine based on liquid chromatography-tandem mass spectrometry. The liquid chromatography-electrospray ionization-tandem mass spectrometry method for quantitation of BPA and BPA-gluc uses stable isotope-labeled internal standards. A linear ion trap mass spectrometer permits identification and quantitation of BPA-gluc and BPA without sample workup. Development of separation conditions reduced the BPA-background in solvent samples to below 2.5 pmol/ml for BPA. Limit of quantitation (LOQ) for BPA in control urine was 15 pmol/ml; LOQ for BPA-gluc was 65 pmol/ml. Application of the method to urine samples from human subjects (n = 6) after administration of 25 microg of BPA/person (estimated maximum human daily intake) permitted the determination of excretion kinetics for BPA-gluc; BPA was below the LOD in all except two of the samples. In urine or blood samples of human subjects (n = 19) without intentional exposure to BPA, BPA concentrations were always below the limit of detection ( approximately 2.5 pmol/ml) with or without prior glucuronidase treatment. The results show that care is required for analysis of BPA and its major metabolite BPA-gluc. The LOD obtained and the absence of detectable levels of BPA in samples from individuals suggests that general exposure of humans to BPA is much lower than the worst-case exposure scenario developed.     

A time-resolved fluorescence immunoassay for the determination of a novel respiratory therapeutic agent, AR-C68397XX (Viozan) in human plasma

A dissociation-enhanced lathanide fluorescence immunoassay (DELFIA™) method has been developed for the determination of AR-C68397XX, a novel respiratory therapeutic agent, in human plasma. The method is a ‘direct’ immunoassay and provides an alternative to the solid phase extraction RIA described in a previous publication, which employs the same specific antiserum. The DELFIA method is suitable for the determination of the analyte at pg ml⁻¹ concentrations. The non-isotopic label was prepared by complexation of a DTPA derivative of AR-C68397XX with free europium cation (Eu³⁺). Plasma samples were diluted at least 5-fold prior to analysis to eliminate matrix interference. The calibration range is 10–2000 pg ml⁻¹, and the LOQ of the method is 50 pg ml⁻¹ using 50 μl of diluted human plasma sample.     

Determination of fentanyl in human plasma by head-space solid-phase microextraction and gas chromatography-mass spectrometry

A head-space solid-phase microextraction (HS-SPME) method coupled to GC–MS was developed to extract fentanyl from human plasma. The protein binding was reduced by acidification and, eventually, the sample was deproteinized with trichloroacetic acid. The parameters influencing adsorption (extraction time, temperature, pH and salt addition) and desorption (desorption time and temperature) of the analyte on the fibre were investigated and validated for method development. The developed method proved to be rapid, simple, easy and inexpensive and offers high sensitivity and reproducibility. Linear range was obtained from 0.1 ng/ml to 2 μg/ml. The limit of detection was 0.03 ng/ml while an inter-day precision of less than 5% (n = 15) could be achieved. The method has been applied for the determination of fentanyl in plasma samples after application of 50 μg/h Duragesic fentanyl patch.     

Capillary zone electrophoresis determination of meropenem in biological media using a high sensitivity cell

A capillary electrophoresis method with a high sensitivity cell (Z-cell) has been developed for the determination of meropenem in aqueous solution and in biological media (urine, plasma). Water samples were analysed using two calibration curves of meropenem with standard capillary and a capillary with a high sensitivity cell. In urine, the samples were only diluted in buffer and were injected without any further sample preparation. For the analysis of plasma samples, a calibration curve was utilized covering the meropenem concentration range of 0.5–200 μg/ml. The detection limit and the relative standard deviation of the migration times and of the peak areas were determined.     

LC determination of enrofloxacin

A simple, rapid and sensitive high-performance liquid chromatographic (HPLC) method was developed for the assay of enrofloxacin in raw material and injection. The validation method yielded good results and included the range, linearity, precision, accuracy, specificity, recovery, limit of detection (LOD) and limit quantification (LOQ) values. The HPLC separation was carried out by reversed phase chromatography on a C-18 absorbosphere column (150×4.6 mm i.d. 5 μm particle size) with a phase composed of sodium acetate (pH 4.7; 0.1 M): acetonitrile (60:40, v/v; pH 5.0), pumped isocratically at a flow rate of 1.5 ml min⁻¹. The effluent was monitored at 278 nm with the eluting solvent. The calibration graph for enrofloxacin was linear from 10.0 to 80.0 μg ml⁻¹.     

Reverse Phase High Performance Liquid Chromatographic Method for the Analysis of Letrozole in Pharmaceutical Dosage Forms

A rapid and sensitive reverse phase high performance liquid chromatographic method is depicted for the qualitative and quantitative assay of letrozole in pharmaceutical dosage forms. Letrozole was chromatographed on a reverse phase C18 column with a mobile phase consisting of acetonitrile and phosphate buffer (pH 7.8) in the ratio of 70:30 v/v. The mobile phase was pumped at a flow rate of 1 ml/min. Acenaphthene was used as an internal standard and the eluents were monitored at 232 nm. The retention time of the drug was 3.385 min. With this method, linearity was observed in the range of 10-100 μg/ml. The LOD and LOQ were found to be 0.51 μg/ml and 1.52 μg/ml, respectively. The method was found to be applicable for analysis of drug in tablets. The results of the analysis were validated statistically.     

Simultaneous Derivative Spectrophotometric Analysis of Doxylamine Succinate, Pyridoxine Hydrochloride and Folic Acid in Combined Dosage Forms

Two UV spectrophotometric methods have been developed, based on first derivative spectrophotometry for simultaneous estimation of doxylamine succinate, pyridoxine hydrochloride, and folic acid in tablet formulations. In method I, the concentrations of these drugs were determined by using linear regression equation. Method II is also based on first derivative spectrophotometry however simultaneous equations (Vierdot''s method) were derived on derivative spectra. The first derivative amplitudes at 270.0, 332.8 and 309.2 nm were utilized for simultaneous estimation of these drugs respectively by both methods. In both the methods, linearity was obtained in the concentration range 2.5-50 μg/ml, 1-40 μg/ml and 1-30 μg/ml for doxylamine succinate, pyridoxine hydrochloride, and folic acid respectively. The developed methods show best results in terms of linearity, accuracy, precision, LOD, LOQ and ruggedness for standard laboratory mixtures of pure drugs and marketed formulations. The common excipients and additives did not interfere in their determinations.     

Comparative evaluation between capillary electrophoresis and high-performance liquid chromatography for the analysis of florfenicol in plasma

A capillary electrophoresis (CE) and a reversed phase high-performance liquid chromatography (RP-HPLC) method with UV detection have been developed for florfenicol analysis in plasma samples. The suitabilities of both methods for quantitative determination of florfenicol were approved through validation specification, such as linearity, precision, selectivity, accuracy, limit of detection and quantification. The capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) assay were compared by analyzing a series of plasma samples containing florfenicol in different concentrations using the two methods. The extraction procedure is simple and no gradient elution or derivatization is required. Furthermore, the analysis time of the CE method is two times shorter than the respective parameter in HPLC and solvent consumptions is considerably lower. The calibration curve were linear to at least 0.05–10 μg/ml (r = 0.9998) and 0.1–10 μg/ml (r = 0.9998) for CE and HPLC, respectively. The separation efficiency are good for both methods. The detection limits for florfenicol were 0.015 μg/ml with CE and 0.03 μg/ml with HPLC and CE method gave lower value, even though UV detector was applied in the both cases. The both methods were selective, robust and reliable quantification of florfenicol and can be useful for clinical and biomedical investigations.     

Determination of gabapentin in human plasma and urine by high-performance liquid chromatography with UV-vis detection

A simple and reliable high-performance liquid chromatographic (HPLC) method with UV–vis detection has been developed and validated for the determination of gabapentin (GBP) in human plasma and urine. The clean up of the sample was carried out by solid-phase extraction with C18-cartridge. After the clean up procedure, the samples were pre-column derivatizated with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS). A chromatographic separation was achieved on a C18 column with a mobile phase consisting of acetonitrile and 10 mM orthophosphoric acid (pH 2.5) with isocratic elution (35:65). Baclofen was used as an internal standard (I.S.). The method developed for GBP was linear over the concentration range of 0.05–5.0 μg/ml and 0.1–10.0 μg/ml for plasma and urine, respectively. The method is precise (relative standard deviation, R.S.D. <4.05%) and accurate (relative mean error, RME <0.15%); mean absolute recoveries were 72.21% for plasma and 72.73% for urine.     

Pharmacokinetic study of a new angiotensin-AT1 antagonist by HPLC

Recently an innovative novel class angiotensin-AT(1) antagonist has been developed by Rottapharm. In this study, we present a validated method for detecting CR 3834 in biological matrices using high-performance liquid chromatography (HPLC) with diode array detection. After oral administration (30mg/kg) to Wistar rats, the plasma and urine concentrations of CR 3834 and its potential metabolic products were determined. Moreover, the plasmatic time course in rats has been determined after intravenous (IV) administration of CR 3834 (5mg/kg). Biological samples (0.5ml of plasma and 1ml of urine) were purified using solid-phase extraction (SPE) of analytes and the internal standard Idebenone, 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1-4-benzoquinone. A chromatographic separation was performed on an Adsorboshere C18 at 25 degrees C, with a pre-column of the same matrix; the eluent was made up of acetonitrile/acidified water with CF(3)COOH (pH 2.01) in ratio of 75:25 (v/v); the flow rate was 1.0ml/min and a 100mul loop. The lower limit of detection (LOD) was taken as 25ng/ml in plasma and 50ng/ml in urine samples. The lower limit of quantification (LOQ) was taken as 0.1 and 0.2mug/ml in plasma and urine samples, respectively. The procedures were validated according to international standards with a good reproducibility and linear response (r=0.9916 in plasma; r=0.9997 in urine). The coefficients of variation inter assay ranged between 2.579 and 4.951% in plasma, and between 0.813 and 2.460% in urine. Mean recovery for CR 3834 was 79% in plasma and 97% in urine samples. The experiments performed demonstrated that the method presented was suitable for determining this new angiotensin-AT(1) antagonist in rat plasma and urine.     

Estimation of Amlodipine Besylate, Valsartan and Hydrochlorothiazide in Bulk Mixture and Tablet by UV Spectrophotometry

A simple, precise, accurate and economic simultaneous UV spectrophotometric method has been developed for the estimation of amlodipine besylate, valsartan and hydrochlorothiazide in combination in bulk mixture and tablet. The estimation was based upon measurement of absorbance at absorbance maxima of 359 nm, 317 nm and 250 nm for amlodipine besylate, hydrochlorothiazide and valsartan in methanol, respectively in bulk mixture and tablet. The Beer Lambert''s law obeyed in the concentration range 5-25 μg/ml, 10-50 μg/ml and 5-25 μg/ml for amlodipine besylate, hydrochlorothiazide and valsartan, respectively. The estimation of bulk mixture and tablet was carried out by simultaneous equation, Q-analysis and area under curve method for estimation of amlodipine besylate and hydrochlorothiazide and standard curve method for estimation of valsartan. The results were found to be in the range of 99.6±1.52% to 102±0.51%. Method was validated with respect to specificity, linearity, range, accuracy, precision, LOD, LOQ, robustness, ruggedness and can be applied for routine analysis of tablet dosage forms.     

Development and application of a radioreceptor assay for scopolamine

6 beta,7 beta-Epoxy-3a(1aH,5aH)-tropanyl-(S)-tropate (scopolamine) has proved to be a very effective drug in the prevention of motion sickness, however, the drug has a small therapeutic window, a low bioavailability and a short half-life. A transdermal drug delivery system (Scopoderm TTS) was developed to circumvent these problems as well as the variability in gastric absorption. In order to study the pharmacokinetics of the drug and its glucuronide, a highly sensitive radioreceptor assay with an absolute detection limit of 15 pg scopolamine was developed. In children undergoing minor surgery, a Scopoderm TTS patch of different sizes (according to the age of the children) was applied to the retro-auricular skin. Urine samples were collected and assayed for free and conjugated scopolamine. Furthermore possible anticholinergic effects on the pupil reaction, salivation, blood pressure and heart rate were monitored. The urine excretion of free and glucuronidated scopolamine showed large intra- and interindividual variations. However, in all age groups relatively high percentages of free scopolamine were found, namely 47.5% (3-6 years), 48.3% (7-12 years) and 36.0% (13-18 years) of the sum of free plus glucuronidated scopolamine. During the application of the patch, a prolonged plateau of scopolamine excretion could be found. Although in general the patches were well tolerated, both locally and systematically, moderate anticholinergic effects were observed in patients.     

Validated HPLC method for determination of LASSBio-581, a new heterocyclic N-phenylpiperazine derivative, in rat plasma

A rapid, simple and accurate high performance liquid chromatography (HPLC) method was developed and validated for the determination of LASSBio-581 (1-[1-(4-chloro-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-4-phenyl-piperazine) in rat plasma using ketoconazole as internal standard. Plasma samples were deproteinized with methanol. A good chromatographic separation was achieved using a reversed phase C₁₈ column. Mobile phase consisting of sodium dihydrogen phosphate monohydrate (pH 4.5, 0.02 M) and methanol mixture (35:65, v/v) was used at a flow rate of 1.0 ml/min. The eluate was monitored using a UV detector at 248 nm. The retention times of LASSBio-581 and the internal standard were approximately 3.8 and 5.6 min, respectively. The calibration curves were linear over the concentration range of 0.25–8.0 μg/ml with correlation coefficients >0.99. The limit of quantitation was 0.25 μg/ml. The accuracy of the method was >90%. The intra-day relative standard deviation (R.S.D.) ranged from 6.15 to 10.52% at 0.4 μg/ml, 7.44 to 13.81% at 1.5 μg/ml and 6.10 to 13.94% at 6.0 μg/ml. The inter-day R.S.D. were 9.54, 8.42 and 8.25% at 0.4, 1.5 and 6.0 μg/ml, respectively. No interference from endogenous substances or metabolites were observed. The method has been used to measure plasma concentrations of LASSBio-581 in pharmacokinetic studies in rats.     

Sensitive SPE-HPLC method to determine a novel angiotensin-AT1 antagonist in biological samples

A high-performance liquid chromatography (HPLC)-method after solid-phase extraction (SPE) has been developed in order to determine a new angiotensin-AT1 antagonist, i.e. CR 3210 (C27H24N8; MW = 460.54), 4-[4-[(2-ethyl-5,7-dimethylimidazo[4,5-b]pyridin-3-yl)methyl]phenyl]-3-(2H-tetrazol-5-yl)quinoline in rat plasma and urine after oral administration to Sprague-Dawley rats. CR 3210 and the internal standard (IS) CR 1505 (loxiglumide), i.e. 4-[(3,4-dichlorobenzoyl)amino]-5-[(3-methoxypropyl)pentylamino]-5-oxopentanoic acid, were isolated from rat urine and plasma by solid-phase extraction. The procedure was optimized regarding the sorbent extraction material, the pH in the conditioning solution, the washing step, the dry time and the type of elution solvent. The separation was performed by reversed-phase high-performance liquid chromatography with ultraviolet detection. The samples were injected onto the analytical column (Tracer Extrasil ODS1) and detected at 238 nm, giving a capacity factor of 1.87 for CR 3210 and 1.10 for the internal standard. The selectivity of the method was satisfactory. The mean recovery of CR 3210 from spiked rat plasma was 68.5 at 75 ng/ml and 80.9 at 3000 ng/ml; the mean recovery of CR 3210 from spiked rat urine was 69.9 at 75 ng/ml and 78.6 at 3000 ng/ml. The lower limit of detection (LOD) was 14 ng/ml in plasma and 22 ng/ml in urine samples. The lower limit of quantification (LOQ) was taken as 30 ng/ml, the lowest calibration standard using 500 microl rat plasma and urine. The procedures were validated according to international standards with a good reproducibility and linear response from 30 to 3000 ng/ml, for either plasma or urine. The sensitivity of the method allowed for its application to pharmacokinetic studies.     

Determination of fentanyl in human plasma and fentanyl and norfentanyl in human urine using LC-MS/MS

Fentanyl, a potent analgesic drug, has traditionally been used intravenously in surgical or diagnostic operations. Formulations with fentanyl in oral transmucosal delivery system and in transdermal depot-patch have also been developed against breakthrough pain in cancer patients. In this report, LC–MS/MS methods to determine fentanyl in human plasma as well as fentanyl and its main metabolite, norfentanyl, in human urine are presented together with validation data. The validation ranges were 0.020–10.0 and 0.100–50.0 ng/ml for fentanyl in plasma and urine, respectively, and 0.102–153 ng/ml for norfentanyl in urine.

Liquid–liquid extraction of the compounds fentanyl, norfentanyl and the deuterated internal standards, fentanyl-d₅ and norfentanyl-d₅ from the matrixes was applied and separation was performed on a reversed phase YMC Pro C₁₈-column followed by MS/MS detection with electrospray in positive mode. The inter-assay precision (CV%) was better than 4.8% for fentanyl in plasma and 6.2% and 4.7% for fentanyl and norfentanyl, respectively, in urine.

The ruggedness of the methods, selectivity, recovery, effect of dilution and long-term stability of the analytes in plasma and urine were investigated. Effect of haemolysis and stability of fentanyl in blood samples were also studied.

The methods have been applied for the determination of fentanyl in plasma samples and fentanyl/norfentanyl in urine samples taken for pharmacokinetic evaluation after a single intra-venous (i.v.) dose of 75 μg fentanyl.     

Pharmacokinetics and Oral Bioavailability of Scopolamine in Normal Subjects

The pharmacokinetics and bioavailability of scopolamine were evaluated in six healthy male subjects receiving 0.4 mg of the drug by either oral or intravenous administration. Plasma and urine samples were analyzed using a radioreceptor binding assay. After iv administration, scopolamine concentrations in the plasma declined in a biexponential fashion, with a rapid distribution phase and a comparatively slow elimination phase. Mean and SE values for volume of distribution, systemic clearance, and renal clearance were 1.4 ± 0.3 liters/kg, 65.3 ± 5.2 liters/hr, and 4.2 ± 1.4 liters/hr, respectively. Mean peak plasma concentrations were 2909.8 ± 240.9 pg/ml following iv administration and 528.6 ± 109.4 pg/ml following oral administration. Elimination half-life of the drug was 4.5 ± 1.7 hr. Bioavailability of the oral dose was variable among subjects, ranging between 10.7 and 48.2%. The variability in absorption and poor bioavailability of oral scopolamine indicate that this route of administration may not be reliable and effective.     

Rapid on-line extraction and quantification of escitalopram from urine using sol-gel columns and mass spectrometric detection

A fast and easy way to quantify escitalopram in urine has been developed. A capillary with a silica based monolithic bed inside is used to extract escitalopram from urine and the for mass spectrometry detrimental matrix is washed away by applied pressure. The analyte is eluted by a solution containing organic modifier and directly electrosprayed into a time-of-flight mass spectrometer, ESI-TOF-MS. This method makes it possible to load large volumes of sample onto the column and preconcentrate escitalopram on-line before detection. Standard addition of escitalopram to the urine sample gave a linear calibration curve (R(2)=0.988). The analyzed sample was found to contain an escitalopram concentration of 0.62 ng/ml, well in line with earlier publications. The calculated LOD was 10 pg/ml and LOQ was 34 pg/ml as compared to earlier reports with a LLOQ of 1 ng/ml. The intra day variation of the escitalopram peak area is less than 6.3%.     

A Rapid LC-HRMS Method for the Determination of Domoic Acid in Urine Using a Self-Assembly Pipette Tip Solid-Phase Extraction

In this study, we developed a self-assembly pipette tip solid-phase extraction (PTSPE) method using a high molecular weight polymer material (PAX) as the adsorbent for the determination of domoic acid (DA) in human urine samples by liquid chromatography high-resolution mass spectrometry (LC-HRMS) analysis. The PTSPE cartridge, assembled by packing 9.1 mg of PAX as sorbent into a 200 μL pipette tip, showed high adsorption capacity for DA owing to the strong cationic properties of PAX. Compared with conventional SPE, the PTSPE is simple and fast, and shows some advantages in the aspects of less solvent consumption, low cost, the absence of the evaporation step, and short time requirement. All the parameters influencing the extraction efficiency such as pH, the amount of sorbent, the number of aspirating/dispensing cycles, and the type and volume of eluent in PTSPE were carefully investigated and optimized. Under the optimized conditions, the limit of detection (LOD) and limit of quantification (LOQ) values of DA were 0.12 μg/L and 0.37 μg/L respectively. The extraction recoveries of DA from the urine samples spiked at four different concentrations were in a range from 88.4% to 102.5%. The intra- and inter-day precisions varied from 2.1% to 7.6% and from 2.6% to 12.7%, respectively. The accuracy ranged from −1.9% to −7.4%.