Sensitive Liquid Chromatographic Assay Research Articles

Development and validation of RP-HPLC method for estimation of anti- hypothyroidism drug in capsule dosage form

A simple, accurate, sensitive and precise Reverse Phase-High Performance Liquid Chromatographic assay method for estimation of Levothyroxine sodium in dosage form was successfully developed. The chromatographic separation was performed on Welch Ultisil XB-CN, 150 x 4.6 mm, 5µm. column. The mobile phase consists of 0.05% OPA Buffer solution, and Acetonitrile in the ratio of 72:28, v/v. Mobile phase was delivered at a flow rate of 1.0 ml/min. Samples were injected 50 μL the column temperature was kept at 40 0C and sample temperature 150C. The wavelength 221 nm were selected for the evaluation of the chromatogram. The retention time of the drug was found to be 3.7 min. The developed method was found to be linear in a concentration range of 5-15 μg/ml of the drug (r2= 0.999). The low value of % RSD indicates reproducibility of the method. Thus this method can be used for routine analysis of pomalidomide in formulation and to check the stability of bulk samples.

HPLC with fluorescence detection for the bioanalysis and pharmacokinetic study of Doxorubicin and Prodigiosin loaded on eco-friendly casein nanomicelles in rat plasma

A rapid, efficient, and sensitive liquid chromatographic assay hyphenated to fluorometric detector (HPLC-FLD) was developed and validated for the determination of doxorubicin (DXR) and prodigiosin (PDG) in rat plasma. The sample pre-treatment involves a protein precipitation with acetonitrile with satisfying extraction efficiency (98% and 85% for DXR and PDG, respectively). The chromatographic separation was accomplished using stationary phase: Agilent Zorbax Eclipse plus-C18 analytical column (250 × 4.6 mm, 5 μm) and gradient eluting mobile phase of ammonium acetate (pH = 3), acetonitrile and methanol with programmed fluorescence detection. As the proposed method has been validated, it was subsequently implemented to evaluate DXR and PDG loaded on novel eco-friendly Casein nano drug delivery system after intravenous injection in healthy rats. A comparative pharmacokinetics’ study was carried out in rats for DXR in free form, DXR alone entrapped in the nanomicelle and DXR with PDG entrapped in the nano micelle. After testing the differences in pharmacokinetic parameters of the different formulations using ANOVA, the results showed insignificant differences among the tested parameters. This indicates that the presented nanomicelle delivery system has succeeded to incorporate PDG and DXR in a hydrophilic, safe, and potent formulation. This novel nanomicelle has negligible effect on the distribution and elimination of DXR.

Rapid and sensitive determination of levofloxacin in microsamples of human plasma by high‐performance liquid chromatography and its application in a pharmacokinetic study

A rapid, sensitive and simple high-performance liquid chromatographic assay with ultraviolet detection was developed for the quantification of levofloxacin in microsamples (100 μL) of human plasma. The extraction procedure included a protein precipitation technique and a short chromatographic running time (4.5 min). Analyses were carried out on a Symmetry C18 column using a mixture of acetonitrile and 0.01 m potassium dihydrogen aqueous solution (pH 3.4; 14:86 v/v) as mobile phase. The method provided specificity and was linear (r ≥ 0.9992) over the concentration range 0.1-12 µg/mL. The average absolute recovery was 93.59%. The intra- and inter-day coefficients of variation were <6%. Additionally, levofloxacin was stable in all evaluations. The usefulness of this method was demonstrated in a pharmacokinetic study of levofloxacin in healthy adult volunteers. The present method offers two main advantages: (a) the use of microsamples reduces the total volume of blood to be collected from patients; and (b) it provides a good cost-effectiveness ratio. It is concluded that the method is rapid, simple, sensitive, economical and suitable for the determination of levofloxacin in human plasma using a small volume of sample.

Development and Validation of a Stability- Indicating High Pressure Liquid Chromatography Method for Determination of Prostaglandin E1 and its Degradation Products in sn Intracavernous Formulation

A new intracavernous formulation to overcome the failure of standard treatments of erectile dysfunction after radical prostatectomy was developed. This formulation contains prostaglandin E1 (pGE1) at 15 μg/ml in association with papaverin and urapidil at 15 mg/ml and 2.5 mg/ml respectively. Since pGE1 was the main compound to be subject to physico-chemical degradation, its degradation and the concomitant apparition of its degradation product prostaglandin A1 (pGA1) are correlated to the stability of the formulation. The low specific absorbance of PGE1 in the ultraviolet region, and the presence of high levels of papaverin and urapidil in the formulation were the principal difficulties to develop a new method for simultaneous determination of pGE1 and pGA1. Many compositions and pH of mobile phase were studied to find the best chromatographic conditions, and the chosen method is a sensitive, precise and accurate ramp reversed-phase high-performance liquid chromatographic (RP-HPLC) assay method. Ramp RP-HPLC separation was achieved on a Kromasil 5 C18 column (250×4.6 mm id, 5 μm particle size) using mobile phase composed of acetonitrile-pH 3 phosphate buffer (37 : 63%, v/v). The detection was performed at 205 and 230 nm for PGE1 and pGA1 respectively, using a multiwave UV detector. The method was validated for specificity, linearity, precision, accuracy, robustness and sensibility. Limits of quantitation were about 3 μg/ml for pGE1 and 0.5 μg/ml for pGA1. The presence of urapidil and papaverin at high concentration did not interfere with determination of pGE1 and pGA1 in the formulation. The method developed which separates all the most degradation products formed under variety of conditions was sensitive, selective, linear, precise and accurate, and thus can be used to study the stability of the formulation.

Validation of a HPLC method for the measurement of erythrocyte encapsulated thymidine phosphorylase (EE-TP) activity

A sensitive and simple reverse-phase high performance liquid chromatographic (HPLC) assay has been validated for the determination of thymine as a measure of thymidine phosphorylase activity encapsulated in erythrocytes (EE-TP), a formulation which is under clinical development as an enzyme replacement therapy for the treatment of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Diluted erythrocyte lysates were incubated in 100mM sodium phosphate buffer and 10mM thymidine at 37°C for 10min and the reaction stopped with 40% trichloroacetic acid. Following centrifugation, the supernatant was washed with water saturated diethyl ether, and injected onto a Spherisorb C18 column (125mm×4.6mm, 5μm), with a mobile phase (40mM ammonium acetate, 5mM tetrabutyl ammonium hydrogen sulphate, pH 2.70) delivered at a flow rate of 1.0ml/min and run time of 8min. Ultraviolet detection (UV) was employed at 254nm. The method was linear in the range of 5–500nmol/ml (r2=0.992), specific with intra- and inter-day precisions of <9.6 and accuracies within ±20%. Limits of detection and quantification were 1.2nmol/ml and 10nmol/ml, respectively. The method was applied to quantify thymidine phosphorylase activity in samples of in-process controls and batches of EE-TP manufactured for clinical use.

Determination of NVP-BEZ235, a dual PI3K and mTOR inhibitor, in human and mouse plasma and in mouse tissue homogenates by reversed-phase high-performance liquid chromatography with fluorescence detection

NVP-BEZ235 is a novel dual inhibitor of PI3K/mTOR and currently undergoing phase I/II clinical trials for advanced solid tumors. We developed a sensitive and selective reversed-phase high-performance liquid chromatographic (HPLC) assay with fluorometric detection for quantification of NVP-BEZ235 in biological matrices. Liquid–liquid extraction with tert-butyl methyl ether was used for sample pre-treatment, yielding a recovery of >84%. Chromatographic separation of NVP-BEZ235 and the internal standard (IS) NVP-BBD130 was achieved on a GraceSmart C-18 column by isocratic elution with a mobile phase which consisted of acetonitrile, methanol, and milliQ water adjusted with acetic acid to pH 3.7 (20:36:44, v/v/v). Fluorescence detection using excitation and emission wavelengths of 270 and 425nm, respectively, provided a selectivity and sensitivity allowing quantification down to 1ng/ml in human plasma and linear calibration curves within a range of 1–1000ng/ml. The assay was validated for human plasma, mouse plasma and a range of tissues. The accuracy, within-day and between-day precision for all matrices, was within the generally accepted 15% range. NVP-BEZ235 was stable for 72h in pretreated samples in reconstitution mixture (acetonitrile–water (30:70, v/v)), but unstable in mouse tissue homogenates upon repeated freeze–thaw cycles or long term storage (≥24h) at room temperature. A pilot pharmacokinetic study in mice demonstrated the applicability of this method for pharmacokinetic purposes. Overall, this assay is suitable for the pharmacokinetic studies of NVP-BEZ235 in mice and in human plasma.

A Stability Indicating UPLC Method for Candesartan in Bulk Drug Samples

A simple, sensitive gradient rapid resolution liquid chromatographic assay method has been developed for the quantitative determination of Candesartan Cilexetil in bulk active pharmaceutical ingredient, used for the treatment of hypertension. The developed method is also applicable for the process related impurities determination. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination delivered in a gradient mode and quantification was by ultraviolet detection at 210 nm at a flow rate of 0.4 mL × min﹣1. In the developed UPLC method the resolution between Candesartan Cilexetil and its two potential impurities was found to be greater than 2.0. Regression analysis showed an r value (correlation coefficient) greater than 0.99 for Candesartan Cilexetil and its two impurities. This method was capable to detect two impurities of Candesartan Cilexetil at a level of 0.003% with respect to test concentration of 1.0 mg × mL﹣1 for a 2 μL injection volume. The bulk active pharmaceutical ingredient was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in oxidative stress conditions. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.5%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.

Influence of water deprivation on the disposition of paracetamol.

The effect of acute (96 h) water deprivation on the disposition of paracetamol (acetaminophen) has been examined in male Sprague-Dawley rats. Plasma and urinary concentrations of the drug and its two major metabolites, the glucuronide and sulphate, were determined by a sensitive and specific high performance liquid chromatographic assay. Following an intravenous dose of 100 mg kg-1 of paracetamol, no significant changes were found in the elimination rate constant (k), the mean residence time (MRT), total plasma clearance (Cl) and the apparent volume of distribution at steady-state (Vss). However, rats deprived of water for 96 h excreted a larger percentage of the administered dose as the glucuronide conjugate (15.3 vs 7.9%) and a smaller percentage as unchanged paracetamol (7.3 vs 20.7%) in the urine. In addition, there was a significant two-fold increase in the partial metabolic clearance to paracetamol glucuronide. Water deprivation also led to a significant reduction in the renal clearance of paracetamol accompanied by an increase in the renal clearance of the glucuronide.

A sensitive high performance liquid chromatographic assay of a new metabolite of ampicillin in man.

A sensitive, high-performance liquid chromatographic method has been developed for the determination of piperazine-2,5-dione, a new metabolite of ampicillin, in human urine. Piperazine-2,5-dione was separated from human urine on a C18-column using phosphate buffer-methanol (pH 3.5) as eluent. Subsequently, the effluent underwent a postcolumn reaction with mercurous chloride and was then detected at 305 nm. It was found that piperazine-2,5-dione was also excreted in the urine of a volunteer dosed with ampicillin.

Pharmacokinetics of Cyanamide in Dog and Rat

A pharmacokinetic study of cyanamide, an inhibitor of aldehyde dehydrogenase (E.C. 1.2.1.3) has been made in the beagle dog and Sprague-Dawley rat. Cyanamide plasma levels were determined by a sensitive high performance liquid chromatographic assay, specific for cyanamide. In the dog, i.v. administration of cyanamide at 1, 2 and 4 mg kg-1, produced a dose-dependent pharmacokinetic behaviour. Statistically significant changes were observed in plasma clearance values (12.6 to 19.7 mL kg-1 min-1), half life values (39 to 61 min) and mean residence times (50 to 79 min). Peak plasma concentrations, after oral administration of 4 mg kg-1 were achieved at 30 min and oral bioavailability was about 65%. In the rat after i.v. or oral administration, cyanamide (2 mg kg-1) had a half life of 30 min, a total plasma clearance of 117 mL kg-1 min-1 and a mean residence time of 26 min. Oral bioavailability was about 69%.

High-performance Liquid Chromatographic Method with Diode Array Detection for Quantification of Haloperidol Levels in Schizophrenic Patients During Routine Clinical Practice

Rationale: Till date, very few HPLC methods are available with short run time for monitoring of haloperidol, facilitating large number of sample analysis within short time frame. Objective: A selective and sensitive reverse phase high-performance liquid chromatographic assay has been developed for monitoring of Haloperidol levels. Methodology: Chromatogram separation of Haloperidol and Loratidine (Internal Standard) was achieved using C18 column as stationary phase. Mobile phase consists of Acetonitrile and Water (50:50), pH-2.5 with 0.1% Acetic Acid and 0.05 M KHPO4 at a flow rate of 1.6 mL min-1. Detection was carried out at 240 nm using UV-PDA detector. Retention time for Haloperidol and Internal Standard was found to be 2:13 and 3:16 minutes respectively. The method has been validated for linearity, specificity, robustness, stability, accuracy and precision. Results: Linearity for Haloperidol was in the range of 03-200 ng mL-1. The total run time of analysis was 5 minutes and the lower limits of detection and quantification were 1.0 and 3.0 ng mL-1 respectively. In present study, most of the enrolled patients were clinically stable on Haloperidol (as evident from various rating scales applied during the study period) at therapeutic range of 5-19 ng mL-1 [13.04 ± 4.03] Discussion: This validated high-performance liquid chromatographic method using a simple mobile phase has been successfully applied for clinical monitoring of Haloperidol in psychiatric patients. The method is economic and its time sparing ability using simple RP-HPLC speaks its utility in clinical and toxicological management over other analytical methods.

Determination of Adenosine and Inosine in Sheep Plasma Using Solid Phase Extraction Followed by Liquid Chromatography with UV Detection

A simple and sensitive liquid chromatography assay following solid phase extraction was developed for simultaneous determination of adenosine and inosine in sheep plasma. The system consisted of a Symmetry C18 column, a mobile phase composed of acetonitrile, 100 mM sodium dihydrogen phosphate and water, and ultraviolet detection at 254 nm. The method showed good sensitivity (limits of detection for adenosine and inosine were 30 and 50 ng/ml, respectively, in the plasma samples), repeatability, and linearity. The developed method was applied to sheep plasma samples from a study examining the cardio active potential of the combination of adenosine and inosine.

Development and Validation of a New Analytical Method for the Determination of Related Components in Quetiapine Hemifumarate

A simple, sensitive isocratic rapid resolution liquid chromatographic assay method has been developed for the quantitative determination of quetiapine hemifumarate in bulk active pharmaceutical ingredient, used for the treatment of schizophrenia. The developed method is also applicable for the process related impurities determination. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination delivered in a isocratic mode and quantification was by ultraviolet detection at 225 nm at a flow rate of 1.0 mL min−1. In the developed LC method the resolution between quetiapine hemifumarate and its three potential impurities was found to be greater than 2.0. Regression analysis showed an r value (correlation coefficient) greater than 0.99 for quetiapine hemifumarate and its three impurities. This method was capable to detect all three impurities of quetiapine hemifumarate at a level of 0.003% with respect to test concentration of 1.0 mg mL−1 for a 3 μL injection volume. The bulk active pharmaceutical ingredient was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in oxidative stress conditions. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.5%. The developed RR-LC method was validated with respect to linearity, accuracy, precision and robustness.

Sensitive high performance liquid chromatographic assay for assessment of doxorubicin pharmacokinetics in mouse plasma and tissues

A sensitive high performance liquid chromatography method (HPLC) has been developed for the quantification of doxorubicin in mouse plasma and tissues. Samples of serum or tissue homogenates, 20 μl, were analyzed following a single step protein precipitation using perchloric acid (35%, v/v). Doxorubicin was separated from the internal standard, daunorubicin, on a Zorbax 300SB C 18 column at 35 °C. Mobile phase was comprised of acetonitrile and water (25:75) containing 0.1% triethylamine, and was adjusted to pH 3 with phosphoric acid. Peaks eluting from the column were detected with a fluorescence detector with excitation and emission wavelengths of 480 and 560 nm, respectively. Standard curves were linear in the range 5–1000 ng/ml, and correlation coefficients were typically greater than 0.999. Intra-assay recoveries ranged from 94.7 to 99.9%, and inter-assay recoveries were in the range of 95.2–101%. The associated coefficient of variation (CV) was less than 10% in all cases. The method was successfully applied to investigate doxorubicin plasma pharmacokinetics and tissue distribution in athymic Fox nu mice.

Development of a Sensitive LC Assay with Fluorescence Detection for the Determination of Zearalenone in Rat Serum

A simple and sensitive liquid chromatographic assay with fluorescence detection assay was developed for the determination of zearalenone levels in rat serum. The assay utilized a single liquid–liquid extraction with t-butyl methyl ether and isocratic elution using a mobile phase consisting of acetonitrile and 0.1% triethylamine in distilled water (pH = 6) (50:50, v/v). Linearity was observed over a concentration range from 10 to 1,000 ng mL−1 (r = 0.9995), with the limit of quantification at 10 ng mL−1 with 100 μL of rat serum. The validated assay was applied to a pharmacokinetic study in rats.

Quantitative Determination of Pravastatin in Pharmaceutical Dosage Forms by High-Performance Liquid Chromatography with Ultraviolet Detection

A simple, sensitive and precise high-performance liquid chromatographic formulations assay for pravastatin (PVS) is described. Good chromatographic separation was achieved using a Teknokroma C8 (5 mm, 25 cm × 4.6 mm) column and a mobile phase consisting of 10mMammonium acetate: methanol: triethylamine (40:60:0.17 v/v/v) while at a flow-rate of 1.0 mL min-1. PVS was detected at 239 nm and was eluted 2.15 min after injection. No endogenous substances were found to interfere. No internal standard was required. Linearity range for PVS was 0.4-1000 mg mL-1. The determination of intra- and inter-day precision (RSD) was less than 2.94 and 2.97%, at all concentration levels. Statistical treatment of the experimental results indicates that the method is precise and accurate. The proposed method was applied to the determination of the component in commercial tablets with no interference from the excipients. A comparative study between the suggested procedure and the pharmacopoeial method for this compound in the tablets showed no significant difference between the two methods.

Stress degradation studies on betahistine and development of a validated stability-indicating assay method

The purpose of this work was to study the stability of betahistine (BET) at different stress conditions and to develop a sensitive stability-indicating high-performance liquid chromatographic (HPLC) assay method. The stress conditions applied were including the effect of heat, moisture, acid–base, and ultra-violet (UV) light. Betahistine and its decomposition products were derivatized by reaction with dansyl chloride (Dan-Cl) and analyzed by HPLC equipped with fluorescence detector (FL) set at 336 and 531 nm as excitation and emission wavelengths, respectively. The drug was particularly labile at UV light and oxygen rich media. Two potential degradation products could be separated and identified by spectral methods. The chromatographic method involved Zorbax Eclipse XDB-C 18 column kept at 30 ± 2 °C and a gradient elution with mobile phase composed of acetonitrile and 0.02 mol L −1 sodium acetate. The response factor of dansylated BET monitored by fluorescence detection was 32 times more than its UV response. The calibration curve of BET in bulk form was linear from 0.005 to 4.2 ng μL −1. Intraday and interday precision were less than 0.04% (CV), and accuracy was between 99.2% and 100.9% over 2.0 ng μL −1. The limit of detection was 0.002 ng μL −1. The method was also validated for sample stability during reaction, robustness and selectivity. The method was applied for purity testing of betahistine in tablet form.

A sensitive and selective liquid chromatographic tandem mass spectrometric assay for simultaneous quantification of novel trioxane antimalarials in different biomatrices using sample-pooling approach for high throughput pharmacokinetic studies

In the present studies, to give momentum to traditionally low throughput pharmacokinetic screening, a bioanalytical method based on the concept of sample pooling for simultaneous bioanalysis of multiple compounds is discussed. A sensitive, selective, specific and rapid HPLC/ESI-MS/MS assay method was developed and validated for the simultaneous quantitation of three novel trioxane antimalarials (99–357, 99–408 and 99–411) in rat plasma using trioxane analogue as internal standard. The suitably validated bioanalytical method was then further extrapolated to rabbit and monkey plasma by performing partial validation. Extraction from the plasma involves a simple two-step liquid–liquid extraction with n-hexane. The analytes were chromatographed on a cyano column by isocratic elution with acetonitrile:ammonium acetate buffer (pH 6) (85:15, v/v) and analyzed by mass spectrometry in multiple reaction-monitoring (MRM) mode. The chromatographic run time was 5.5 min and the weighted (1/ x 2) calibration curves were linear over a range of 1.56–200 ng/ml. The limit of detection (LOD) and lower limit of quantification (LLOQ) in rat plasma, rabbit plasma and monkey plasma were 0.78 and 1.56 ng/ml, respectively, for all three analytes. The intra- and inter-batch accuracy and precision in terms of % bias and % relative standard deviation were found to be well within the acceptable limits (<15%). The average absolute recoveries of 99–357, 99–408 and 99–411 from spiked plasma samples were >90%, >70% and >60%, respectively. The assay method described here could be applied to study the pharmacokinetics of 99–357, 99–408 and 99–411 using sample-pooling technique.

Determination of topotecan in human and mouse plasma and in mouse tissue homogenates by reversed‐phase high‐performance liquid chromatography

A sensitive and selective reversed-phase high-performance liquid chromatographic (HPLC) assay has been developed and validated for quantification of total topotecan in human and mouse plasma and in mouse tissue samples. Isocratic separation was achieved on a Zorbax SB-C(18) column and topotecan was monitored fluorimetrically. Two ranges of calibrations curves were used to determine lower levels of topotecan more accurately. Acceptable accuracy and precision was achieved for all matrices. Topotecan was stable upon repeated freeze-thawing for three cycles or storage for 24 h at ambient temperatures in spiked plasma samples and tissue homogenates, except in heart homogenates. In an additional validation experiment in which (14)C-labeled topotecan was administered to mice, the levels of unchanged topotecan were about 80-90% of the total radioactivity in tissue homogenates collected 10 min after drug administration and virtually similar as in plasma samples. However, results in tissue homogenates obtained 4 h post-drug administration indicated substantial metabolism of topotecan. This assay is suitable for studying the pharmacokinetics and tissue distribution of topotecan in mice. Our results demonstrate the importance of including all tissues of interest for pharmacokinetic studies in the validation procedure.

Development and validation of an HPLC method for the determination of dibenzoylmethane in rat plasma and its application to the pharmacokinetic study

A highly sensitive and simple high-performance liquid chromatographic (HPLC) assay has been developed and validated for the quantification of dibenzoylmethane (DBM) in rat plasma. DBM and internal standard (I.S.) 1-(5-chloro-2-hydroxy-4-methylphenyl)-3-phenyl-1,3-propanedione (CHMPP) were extracted from rat plasma by ethyl acetate/methanol (95:5, v/v) and analyzed using reverse-phase gradient elution with a Phenomenex Gemini C18 5-μm column. A gradient of mobile phase (mobile phase A: water/methanol (80:20, v/v) with 0.1% TFA and mobile phase B: acetonitrile with 0.1% TFA) at a flow rate of 0.2 mL/min, and ultraviolet (UV) detection at 335 nm were utilized. The lower limit of quantification (LLOQ) using 50 μL rat plasma was 0.05 μg/mL. The calibration curve was linear over a concentration range of 0.05–20 μg/mL. The mean recoveries were 80.6 ± 5.7, 83.4 ± 1.6 and 77.1 ± 3.4% with quality control (QC) level of 0.05, 1 and 20 μg/mL, respectively. Intra- and inter-day assay accuracy and precision fulfilled US FDA guidance for industry bioanalytical method validation. Stability studies showed that DBM was stable in rat plasma after 4 h incubation at room temperature, one month storage at −80 °C and three freeze/thaw cycles, as well as in reconstitute buffer for 48 h at 4 °C. The utility of the assay was confirmed by the successful analysis of plasma samples from DBM pharmacokinetics studies in the rats after oral and intravenous administrations.