Simple and sensitive high-performance liquid chromatographic method for the determination of an everninomycin,SCH 27899, in rat plasma

A simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of SCH 27899, an everninomycin antibiotic, in rat plasma. The method involved plasma protein precipation with acetonitrile, followed by reversed-phase HPLC analysis using a polymeric column and a mobile phase containing acetonitrile and ammonium phosphate, pH 7.8. The linear relationship between detector response and concentration was demonstrated with a correlation coefficient of larger than 0.996 at concentrations ranging from 0.2 to 100 μg/ml. The results showed that the HPLC method was accurate (bias ≤6%) and precise (coefficient of variation, C.V.≤6%). The limit of quantitation was 0.2 μg/ml with a C.V. of 2.6% and bias of 5%. SCH 27899 was stable in rat plasma at −20°C for at least 40 days. The HPLC method has been utilized for the determination of SCH 27899 in plasma samples from rats following single intravenous administration (3 mg/kg).     

Rapid determination of loratadine in small volume plasma samples by high-performance liquid chromatography with fluorescence detection

A simple and rapid high-performance liquid chromatographic method with fluorescence detection was developed for the determination of loratadine in small volume plasma samples. Liquid-liquid extraction of loratadine and diazepam (as internal standard) from plasma samples was performed with n-butyl alcohol/n-hexane (2:98, v/v) in alkaline condition followed by back-extraction into diluted perchloric acid. Chromatography was carried out using a C8 column (250 x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-20 mM sodium dihydrogen phosphate-triethylamine (43:57:0.02, v/v), pH 2.4. Analyses were run at a flow-rate of 1.0 ml/min at room temperature. The method was specific and sensitive with a quantitation limit of 0.62 ng/ml and a detection limit of 0.2 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery of loratadine from plasma was 84%, while the intra-and inter-day coefficient of variation and percent error values of the assay method were all less than 9.7%. Linearity was assessed in the range of 0.62-20 ng/ml in plasma with a correlation coefficient of greater than 0.999. The method has been used to analyze several hundred human plasma samples for bioavailability studies.     

Determination of plasma cyclic AMP with automated radioimmunoassay system (Gamma-flo)

We have adapted the Gamma-flo automated radioimmunoassay system to analyze for cyclic 3',5'-AMP in plasma. Direct analysis of plasma samples was not possible due to an interfering substance(s) in plasma. Evidence suggested that the interfering substance is human serum albumin. An efficient, relatively simple method was developed by applying the acetylation procedure to ultrafiltrates of plasma samples and utilizing the Gamma-flo system for rapid radioimmunoassay (50 samples per hour) of cyclic AMP content in the filtrates. Coefficient of variation within assays averaged 3% and interassay coefficient of variation was 4.7%. Recovery of cyclic AMP from plasma into the ultrafiltrate ranged from 93 to 100%. Increments of cyclic AMP added to plasma were recovered linearly up to approximately 60 pmol/ml, the highest concentration tested.     

High-performance liquid chromatographic assay for the α-melanotropin[4,10] fragment analogue (Melanotan-II) in rat plasma

A high-performance liquid chromatographic (HPLC) procedure has been developed for the quantification of Melanotan-II (MT-II), a cyclic heptapeptide which promotes rapid tanning of the skin, in rat plasma. The method involves precipitation of plasma proteins followed by direct-injection HPLC with ultraviolet detection. Calibration curves were linear over the range 100–1000 ng/ml for rat plasma. The method is reproducible and reliable with a detection limit of 50 ng/ml in plasma. Within- and between-day precision and accuracy reported as coefficient of variation and relative error, respectively, were < 7%. The application of the assay was successfully demonstrated by quantifying the concentration of MT-II in rat plasma samples following an intravenous dose of 0.3 mg/kg.     

Sensitive determination of clarithromycin in human plasma by high-performance liquid chromatography with spectrophotometric detection

A rapid, selective and sensitive high-performance liquid chromatographic method with spectrophotometric detection was developed for the determination of clarithromycin in human plasma. Liquid-liquid extraction of clarithromycin and norverapamil (as internal standard) from plasma samples was performed with n-hexane/1-butanol (98:2, v/v) in alkaline condition followed by back-extraction into diluted acetic acid. Chromatography was carried out using a CN column (250 mm x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-50 mM aqueous sodium dihydrogen phosphate (32:68, v/v), pH 4.5. Detection was made at 205 nm and analyses were run at a flow-rate of 1.0 ml/min at 40 degrees C. The analysis time was less than 11 min. The method was specific and sensitive with a quantification limit of 31.25 ng/ml and a detection limit of 10 ng/ml in plasma. The mean absolute recovery of clarithromycin from plasma was 95.9%, while the intra- and inter-day coefficient of variation and percent error values of the assay method were all less than 9.5%. Linearity was assessed in the range of 31.25-2000 ng/ml in plasma with a correlation coefficient of greater than 0.999. The method was used to analyze several hundred human plasma samples for bioavailability studies.     

Estimation of carboxylic acid metabolite of clopidogrel in Wistar rat plasma by HPLC and its application to a pharmacokinetic study

A new HPLC method was developed for the estimation of carboxylic acid metabolite of clopidogrel bisulfate in rat plasma using atorvastatin as internal standard. Plasma samples were extracted with a mixture of ethyl acetate and di-chloro methane (80:20, v/v) followed by subsequent reconstitution in a mixture of water:methanol:acetonitrile (40:40:20, v/v). The chromatographic separation was achieved with gradient elution on Kromasil ODS, 250 mm x 4.6 mm i.d., 5 microm analytical column maintained at 30 degrees C. Carboxylic acid metabolite of clopidogrel as well as the internal standard were detected at a wavelength of 220 nm. The method was validated as per USFDA guidelines. Calibration curves were linear in the concentration range of 125.0-32,000 ng/ml and the correlation coefficient was better than 0.999. The extraction efficiency for the carboxylic acid metabolite of clopidogrel was more than 85.76%. The intra-day accuracy ranged from 98.9% to 101.5% with a precision of 1.30% to 6.06%. Similarly, the inter-day accuracy was between 96.2% and 101.1% with a precision of 3.47% to 4.30%. The drug containing plasma samples were stable at -70 degrees C for 48 days and at ambient temperature for 24h. In the auto-sampler maintained at 15 degrees C, the processed and reconstituted samples were stable for 35 h. The drug containing frozen plasma samples were stable enough to with stand three freeze thaw cycles. The method was successfully applied to the pharmacokinetic study of the two different polymorphs of clopidogrel bisulfate in Wistar rat.     

Liquid chromatographic assay for dicloxacillin in plasma

A simple high-performance liquid chromatographic method for the determination of dicloxacillin in plasma has been developed. The method only requires 0.5 ml of plasma, phosphate buffer solution (pH = 4.7), acidification with 0.5N hydrochloride acid and liquid extraction with dichloromethane. Posterior evaporation of organic under nitrogen steam and redissolution in mobile phase is carried out. The analysis was performed on a Spherisorb C18 (5 microm) column, using methanol -0.05 M phosphate buffer, pH = 4.7 (75:25; v/v) as mobile phase, with ultraviolet detection at 220 nm. Results showed that the assay is sensitive: 0.5 microg/ml. The response is linear in the range of 0.5 - 10 microg/ml. Maximum inter-day coefficient of variation was 12.4%. Mean extraction recovery obtained was 96.95%. Stability studies showed that the loss was not higher than 10%, samples are stable at room temperature for 6 h, at -20 Celsius for 2 months, processed samples were stable at least for 24 h and also after two freeze-thaw cycles. The method has been used to perform pharmacokinetic and bioequivalence studies in humans.     

Determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in human and animal plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A highly sensitive HPLC method with automated column switching was developed for the simultaneous determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in plasma samples from man, Cynomolgus monkey, rabbit, rat and mouse. Plasma (0.4 ml) was deproteinated by adding ethanol (1.5 ml) containing the internal standard acitretin. After centrifugation, 1.4 ml of the supernatant were directly injected onto the precolumn packed with LiChrospher 100 RP-18 (5 μm). 1.25% ammonium acetate and acetic acid-ethanol (8:2, v/v) was used as mobile phase during injection and 1% ammonium acetate and 2% acetic acid-ethanol (102:4, v/v) was added, on-line, to decrease the elution strength of the injection solution. After backflush purging of the precolumn, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm. Two coupled Superspher 100 RP-18 endcapped columns (both 250×4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid: (A) 600:300:60:10 (v/v/v/v), (B) 950:20:5:20 (v/v/v/v), and (C) 990:5:0:5 (v/v/v/v). The method was linear in the range 0.3–100 ng/ml, at least, with a quantification limit of 0.3 ng/ml. The mean recoveries from human plasma were 93.2%–94.4% and the mean inter-assay precision was 2.8%–3.2% (range 0.3–100 ng/ml). Similar results were obtained for animal plasma. The analytes were found to be stable in the plasma of all investigated species stored at −20°C for 4.3 months and at −80°C for 9 months, at least. At this temperature, human plasma samples were even stable for 2 years. The method was successfully applied to more than 6000 human and 1000 animal plasma samples from clinical and toxicokinetic studies. Endogenous levels determined in control patients and pregnant women were similar to published data from volunteers.     

Automated solid-phase extraction and liquid chromatographic method for retinoid determination in biological samples

In this study, a method for partly automated sample preparation and fully automated solid-phase extraction method for plasma, kidney and liver samples for various retinoids like all-trans-4-oxo-retinoic acid, 13-cis-4-oxo-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid, all-trans-retinoic acid, retinol and retinyl palmitate was established. Plasma, embryo-, kidney-and liver-homogenates were automatically mixed and extracted on multiple usage solid-phase (C2) extraction cartridges immediately before HPLC analysis. Automated cleaning, preconditioning and incorporation of the loaded cartridge to fully automated HPLC separation and quantification of the various retinoids in a single HPLC run was established. The recovery of the retinoids was generally between 80 and 90%. Intra-day repeatability was < 11.7%. As little as 1.2 ng/ml could be quantified in lipid-mixture standard samples. This method allows a highly automated sample preparation and a fully automated solid-phase extraction with good selectivity for the study of endogenous retinoids and retinoids after nutritional supplementations and pharmacological applications in several biological samples.     

Development and validation of a liquid chromatographic method for Casiopeina IIgly in rat plasma

A sensitive and specific liquid chromatographic method using solid-phase extraction with Sep-pak cartridges has been developed for the determination of Casiopeina IIgly and validated over the linear range 2.5-50 microg/ml in rat plasma. The analysis was performed on a Symetry C(18) (5 microm) column with a Phenomenex C(18) precolumn. The mobile phase was methanol-water (58:42, v/v). The column effluent was monitored at 273 nm. The results showed that the assay is sensitive at 2.5 microg/ml. Maximum intra-day coefficient of variation was 11.47%. The recovery based upon addition of internal standard to rat plasma was 80.98%. The method was used to perform preclinical pharmacokinetic studies in rat plasma and was found to be satisfactory.     

Specific,sensitive and accurate liquid chromatographic-tandem mass spectrometric method for the measurement of ribavirin in rat and monkey plasma

Ribavirin is a purine nucleoside analog with broad spectrum activity against a spectrum of DNA and RNA viruses. To facilitate pharmacokinetics studies, a LC-MS-MS method for the analysis of ribavirin in rat and monkey plasma was developed and validated. The method involved the addition of acyclovir as an internal standard and protein precipitation with acetonitrile followed by separation by an Intertsil Silica column and quantification by a MS-MS equipped with a positive electrospray ionization in the multiple reaction monitoring mode. The MS-MS reaction was selected to monitor the 245-->113 and 226-->152 transitions for ribavirin and internal standard, respectively. The calibration curve was linear over a concentration range of 10-5000 ng/ml. The lower limit of quantitation was 10 ng/ml, the coefficient of variation (CV) was 8-11%, and the bias was 1-3%. Intra-day and inter-day analysis of QC samples at 30, 1500 and 3500 ng/ml indicate that the method was precise (CV<18%) and accurate (bias<13%). Ribavirin in rat and monkey plasma was stable at 5 degrees C for at least 24 h, 0 degrees C for at least 4 h, and after three freeze-thaw cycles. This specific, accurate and precise assay is useful in the study of the pharmacokinetics of this compound.     

Determination of scopoletin in rat plasma by high performance liquid chromatographic method with UV detection and its application to a pharmacokinetic study

A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed and validated for determination of scopoletin in rat plasma using psoralen as internal standard. Chromatographic separation was achieved on a C(18) column using methanol and distilled water (49:51, v/v) containing 0.05% (v/v) phosphoric acid as mobile phase. The UV detector was set at 345 nm. The calibration curve was linear over the range of 0.165-9.90 microg/ml with a correlation coefficient of 0.9994. The recovery for plasma samples of 0.165, 1.32 and 6.60 microg/ml was 93.2%, 95.9% and 95.5%, respectively. The RSD of intra- and inter-day assay variations was less than 6.7%. This HPLC assay is a precise and reliable method for the analysis of scopoletin in pharmacokinetic studies.     

Development of a liquid chromatographic method for bioanalytical applications with sildenafil

An improved HPLC method was developed for the determination of sildenafil concentrations in plasma. Analysis of sildenafil in plasma samples was simplified by utilizing a one-step liquid-liquid extraction after alkaline treatment of only 1 ml of plasma. The lower limit of quantitation was 10 ng/ml with a coefficient of variation of less than 20%. A linear range was found to exist from 10 to 1000 ng/ml. This HPLC method was validated with precisions (coefficient of variation, C.V.) for inter- and intra-day runs of 0.41-11.15% and 0.36-8.05%, respectively, and accuracies (the relative error of the mean, REM) for inter- and intra-day runs of -8.72-6.81% and 0.41-11.15%, respectively. A bioavailability study of sildenafil was performed on one normal healthy male volunteer by analyzing sildenafil plasma concentrations with this validated HPLC method. Results demonstrated that this HPLC method is appropriate for applications to bioavailability studies of sildenafil. In addition, an example of the influence of the co-administration of grapefruit juice on sildenafil pharmacokinetics in a healthy volunteer is presented.     

Headspace solid-phase microextraction and capillary gas chromatographic-mass spectrometric determination of rivastigmine in canine plasma samples

A simple, rapid and sensitive method for determination of rivastigmine in plasma samples was developed using headspace solid-phase microextraction (HS-SPME) and gas chromatography with mass spectrometry (GC-MS). The optimum conditions for the SPME procedure were: headspace extraction on a 65-microm polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber; 0.5 ml of plasma modified with 1.0 ml of sodium hydroxide-sodium carbonate solution (0.7 M:0.5M); extraction temperature of 100 degrees C, with stirring at 2000 rpm for 30 min. The calibration curve showed linearity in the range from 0.2 to 80 ng/ml with regression coefficient corresponding to 0.9965 and coefficient of the variation of the points of the calibration curve lower than 10%. The quantification limit for rivastigmine in plasma was 0.2 ng/ml. The method was applied to determination of rivastigmine in canine plasma samples from animals after a single oral administration.     

Analysis of betamethasone in rat plasma using automated solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry. Determination of plasma concentrations in rat following oral and intravenous administration

A method is described for the determination of betamethasone in rat plasma by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The analyte was recovered from plasma by solid-phase extraction and subsequently analyzed by LC-MS-MS. A Packard Multiprobe II, an automated liquid handling system, was employed for the preparation and extraction of a 96-well plate containing unknown plasma samples, standards and quality control samples in an automated fashion. Prednisolone, a structurally related steroid, was used as an internal standard. Using the described approach, a limit of quantitation of 2 ng/ml was achieved with a 50 microl aliquot of rat plasma. The described level of sensitivity allowed the determination of betamethasone concentrations and subsequent measurement of kinetic parameters of betamethasone in rat. Combination of automated plasma extraction and the sensitivity and selectivity of LC-MS-MS offers a valuable alternative to the methodologies currently used for the quantitation of steroids in biological fluids.     

Liquid chromatographic method for determination of piperine in rat plasma: application to pharmacokinetics

Piperine, a major alkaloid of Piper longum and Piper nigrum has been reported to have several pharmacological/toxicological effects. Though a number of methods for analysis of this omnipresent food component in pepper fruits are available, its analysis in body fluids has been largely neglected. A high-performance liquid chromatography method for the analysis of piperine in rat plasma is presented in this communication. Analysis was performed using a Symmetry C(18) column (250x4.6 mm) by isocratic elution with 25 mM KH(2)PO(4) (pH 4.5)-acetonitrile (35:65) and UV detection at 340 nm. The calibration plot was linear over the range studied (2-2000 ng) with correlation coefficient of 0.9984. Limit of detection and limit of quantitation were 1 ng/ml and 3 ng/ml, respectively. Good overall recovery (85.5+/-6%) was obtained with 4 ml ethyl acetate and extraction time of 3 min. Intra- and inter-assay coefficient of variation was found to be less than 7.5%. Plasma concentration-time profile of piperine in a conscious rat implanted with jugular vein cannula was obtained using this method. The method is simple, sensitive and reproducible.     

Liquid chromatography coupled with multi-channel electrochemical detection for the determination of daidzin in rat blood sampled by an automated blood sampling system

Daidzin, a soy-derived biologically active natural product, has been reported to inhibit mitochondrial aldehyde dehydrogenase and suppress ethanol intake. This paper describes a method for the determination of daidzin in rat blood. After administration of daidzin, blood samples were periodically collected from awake, freely moving animals by a Culex automated blood sampler. Daidzin was extracted from 50 microl of diluted blood (blood and saline at a ratio of 1:1) with ethyl acetate. Chromatographic separation was achieved within 12 min using a microbore C(18) (100 x 1.0 mm) 3 microm column with a mobile phase containing 20 mM sodium acetate, 0.25 mM EDTA, pH 4.3, 4% methanol and 11% acetonitrile at a flow-rate of 90 microl/min. Detection was attained using a four-channel electrochemical detector with glassy carbon electrodes using oxidation potentials of +1100, 950, 850, 750 mV vs. Ag/AgCl. The limit of detection for daidzin in rat plasma was 5 ng/ml at a signal-to-noise ratio of 3:1. The extraction recovery of daidzin from rat plasma was over 74%. Linearity was obtained for the range of 25-1000 ng/ml. The intra- and inter-assay precisions were in the ranges of 2.7-6.6 and 1.9-3.7%, respectively. This method is suitable to routine in vivo monitoring of daidzin in rat plasma.     

Determination of amitraz in canine plasma by solid-phase microextraction-gas chromatography with thermionic specific detection

A simple and rapid analytical method is presented for the determination of amitraz in canine plasma samples using solid-phase microextraction (SPME) and gas chromatography with thermionic specific detection (GC-TSD). The best conditions for the SPME procedure were: direct extraction on a polydimethlysiloxane (PDMS) fiber with 100-microm film thickness; 400 µl of sample plasma matrix modified with 4 ml sodium borate solution (0.01 mol l(-1), pH 6.5); extraction temperature 70 degrees C, with stirring at 2500 rpm for 45 min. The method was linear between 20 and 400 ng ml(-1) with regression coefficients corresponding to 0.998 and coefficient of the variation of the points of the calibration curve lower than 15%. The lowest limit of quantification (LOQ) for amitraz in plasma was 20 ng ml(-1). This LOQ was determined as the lowest concentration on the calibration curve in which the coefficient of variation was lower than 15%. The proposed method was applied to determine amitraz concentrations in canine plasma to look for toxicity after treatment with amitraz in a dipping bath.     

Application of amino acid analysis by high-performance liquid chromatography with phenyl isothiocyanate derivatization to the rapid determination of free amino acids in biological samples

An amino acid analysis by reversed-phase high-performance liquid chromatography after precolumn derivatization with phenyl isothiocyanate was adapted to the determination of free amino acids in plasma or other biological fluids and in tissue homogenates. Preparation of samples included deproteinization by 3% sulphosalicylic acid, and careful removal under high vacuum of residual phenyl isothiocyanate after derivatization. A Waters Pico-Tag column (15 cm long) was used, immersed in a water-bath at 38°C. In rat or human plasma, separation of 23 individual amino acids, plus the unresolved pair tryptophan and ornithine, was obtained within 13 min. Including the time for column washing and re-equilibration, samples could be chromatographed at 23-min intervals. Variability was tested for each amino acid by calculating the coefficients of variation of retention times (less than 1% in the average) and peak areas (less than 4% for both intra-day and inter-day determinations). The linearity for each standard amino acid was remarkable over the concentration range 3–50 nmol/ml. The mean recovery of amino acid standards added to plasma prior to derivatization was 97 ± 0.8%, except for aspartate (82%) and glutamate (81%). This method is rapid (almost three samples per hour can be analysed, more than in any other reported technique), with satisfactory precision, sensitivity and reproducibility. Therefore, it is well suited for routine analysis of free amino acids in both clinical and research work.     

Liquid chromatography-tandem electrospray mass spectrometry method for determination of serial chiral novel anticholinergic compounds of phencynonate in rat plasma

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of serial chiral novel anticholinergic compounds of phencynonate in rat plasma. After a simple protein-precipitation using methanol, the post-treatment samples were separated on a CAPCELL UG120 column with a mobile phase of a mixture of methanol and water (35:65) containing 0.1% formic acid. The serial chiral analytes and internal standard (IS) were all detected by the use of selected reaction monitoring mode (SRM). The method of all serial chiral analytes developed was validated in rat plasma with a daily working range of 0.5-100 ng/ml with correlation coefficient, R(2) > or = 0.99 and a sensitivity of 0.5 ng/ml as lower limit of quantification, respectively. This method was fully validated for the accuracy, precision and stability studies for all serial chiral analytes. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of serial chiral novel anticholinergic compounds of phencynonate in rat plasma.