Sensitive High-performance Liquid Chromatography Research Articles

High-performance liquid chromatography tandem mass spectrometry for simultaneous detection of aflatoxins B1, B2, G1 and G2 in Indian medicinal herbs using QuEChERS-based extraction procedure

ABSTRACTSince the discovery of aflatoxins (AFs) in the 1960s, much research has focused on detecting the toxins in contaminated food and feedstuffs. But the quality determination in medicinal plant matrices with respect to AFs is scare. Hence, a simple, accurate and sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of AFs AFB1, AFB2, AFG1 and AFG2 in two Indian popular medicinal herbs i.e. senna (Cassia angustifolia) and kalmegh (Andrographis paniculata). AFs have been extracted from herb matrix using a QuEChERS (quick, easy, cheap, effective, rugged and safe)-based extraction procedure followed by applying primary secondary amine and C18 for further clean-up step and then were quantified under the multiple reaction monitoring together with positive ionisation modes. Matrix-matched calibration was used for quantification in order to reduce the matrix effect. Validation of the method was carried out in herbs by recovery experiments. Recoveries of the spiked samples were in the range of 61.9–111.5% with an inter-day and intraday relative standard deviation lower than 20.0%. Limits of detection and quantification ranged from 0.41 to 0.95 ng mL−1 and 1.2 to 3.8 μg kg−1, respectively. The expanded uncertainty of the method was <21% for all the toxins in both the herbs. Finally, the proposed method was successfully applied to determine AF residues in real field samples of senna and kalmegh obtained from different locations in India.

Development and Validation of a Rapid High-Performance Liquid Chromatography⁻Tandem Mass Spectrometric Method for Determination of Folic Acid in Human Plasma.

There are health concerns associated with increased folic acid intake from fortified food and supplements. Existing analytical methods, however, which can be employed to carry out epidemiological and bioavailability studies for folic acid involve laborious sample preparation and/or lengthy chromatographic analysis. In this paper we describe a simple, rapid, and sensitive high-performance liquid chromatography–electrospray ionisation-tandem mass spectrometry (HPLC–ESI-MS/MS) method for determination of unmetabolised folic acid in human plasma using folic acid-d4 as an internal standard. The method required only a simple sample preparation step of protein precipitation and had a total run time of 3.5 min, which is the shortest run time reported to date for HPLC–MS/MS method employed for quantifying folic acid in plasma. The analytes were separated on a C18 column (3 µm; 50 × 3.00 mm) using an isocratic mobile phase consisting of ammonium acetate (1 mM)-acetic acid-acetonitrile (9.9:0.1:90, v/v/v). The method was fully validated in terms of accuracy, precision, linearity, selectivity, recovery, matrix effect, and stability. The short run time and the minimal sample preparation makes the method a valuable tool for performing high-throughput analyses. To demonstrate the applicability of the method in real conditions, it was applied successfully in a bioavailability study for the determination of unmetabolised folic acid levels in vivo in human plasma after oral administration of folic acid.

High-performance liquid chromatography-based assay for glutathione transferase theta 2 activity: Application to characterize interindividual variability in human liver fractions

Human glutathione transferase T2-2 (GSTT2-2) is one of the enzymes considered to play a role in inactivation of toxicants and carcinogens. The expression level of this enzyme is determined by genetic and environmental factors, which may lead to differences in susceptibility. As a specific assay for GSTT2-2 so far a spectroscopical assay based on GSH-conjugation of menaphthyl sulfate (MSu) was used. This spectrophotometric assay, however, appeared too insensitive to accurately quantify the GSTT2-2 activities in a panel of 20 human liver samples. More recently, expression levels of GSTT2-2 in biological samples are quantified by measuring mRNA levels. Since mRNA-levels do not always correlate well with enzyme activity, a specific and sensitive assay is required. In the present study a highly sensitive high-performance liquid chromatography (HPLC)-based method was developed. By applying the new method, firstly, the specificity of GSTT2-2 among 15 recombinant human GST isoforms in catalyzing GSH-conjugation of MSu was confirmed. In addition, a 65-fold inter-individual variation of GSTT2-2 activity was found from the individual liver fractions. By applying the method to individual liver fractions, a 65-fold inter-individual variation of GSTT2-2 activity was found. As a second application, the role of GSTT2-2 in GSH-conjugation of the environmental carcinogen 1-methylpyrene sulfate (MPS) was studied by correlation analysis with GSTT2-2-catalyzed MSu conjugation. The relatively poor correlation suggested that other GSTs also contribute to MPS-conjugation, as confirmed by incubations with recombinant GSTs.

Simultaneous Qualitative and Quantitative Study of Main Compounds in Commelina communis Linn. by UHPLC-Q-TOF-MS-MS and HPLC-ESI-MS-MS.

This study reported the identification and determination of the main components of Commelina communis Linn. A total of 62 compounds were identified in C. communis Linn. extract, which included 29 flavonoids and flavonoid glycosides, 17 phenolic acids, 4 alkaloids, 1 pyrimidine alkaloids, 3 sterols and 8 fatty acids and others by ultra high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry. Moreover, a specific, simple, rapid and sensitive high performance liquid chromatography with tandem mass spectrometry method was developed and validated for determination of 13 components of C. communis Linn., which included orientin, iso-orientin, vitexin, isovitexin, rutin, apigenin, protocatechuate, vanillic acid, caffeic acid, ferulic acid, luteolin, quercetin and isorhamnetin. All calibration curves showed good linearity (r ≥ 0.9991) within the test range. The intra- and inter-day precisions (relative standard deviation%, RSD%) were within 1.04 and 0.92%, and the recoveries ranged from 98.64 to 100.8%. These results may contribute to the further study and quality control for C. communis Linn.

Simultaneous pharmacokinetics and stability studies of physalins in rat plasma and intestinal bacteria culture media using liquid chromatography with mass spectrometry.

Physalins are the major steroidal constituent of Physalis plants and display a range of biological activities. For this study, a rapid and sensitive high-performance liquid chromatography with triple quadrupole mass spectrometry method was developed for the simultaneous quantification of six physalins. Specifically, it was for the quantification of physalin A, physalin B, physalin D, physalin G, 4,7-didehydroneophysalin B, and isophysalin B in rat plasma and rat intestinal bacteria. After a solid-phase extraction, analytes and internal standards (prednisolone) were separated on a Shield reverse-phase C18 column (measuring 3mm×150mm with an internal diameter of 3.5μm) and determined using multiple reactions in a monitoring mode with a positive-ion electrospray ionization source. The mobile phase was a mixture of 0.1% formic acid in water (A) and acetonitrile (B) and was used at a flow rate of 0.6mL/min. The intra- and interday precisions were within 15% with accuracies ranging from 86.2 to 114%. The method was validated and successfully applied to pharmacokinetics and stability studies of six physalins in rat plasma and rat intestinal bacteria, respectively. The results showed that physalin B and isophysalin B could not be absorbed by rats, and rat intestinal bacteria could quickly transform physalins.

A simple and sensitive HPLC–MS/MS method for quantification of eggmanone in rat plasma and its application to pharmacokinetics

Allosteric phosphodiesterase 4 (PDE4) inhibitors are highly sought after due to their important anti-inflammatory and anti-cancer therapeutic effects. We recently identified Eggmanone, an extraordinarily selective allosteric PDE4 inhibitor displaying favorable drug properties. However, a specific analytic method of Eggmanone in serum and its pharmacokinetics have not been reported yet. In this study, we developed a rapid and sensitive high performance liquid chromatography–mass spectrometric (HPLC–MS/MS) method to determine Eggmanone concentrations in rat plasma. This assay method was validated in terms of specificity, linearity, sensitivity, accuracy, precision, matrix effect, recovery and stability, and was applied to a pharmacokinetic study in rats following intravenous injection of Eggmanone at doses of 1 and 3 mg/kg. The lower limit of quantification (LLOQ) of this assay was 5 ng/mL and the linear calibration curve was acquired with R2 > 0.99 between 5 and 1000 ng/m. The intra-day and inter-day precision was evaluated with the coefficient of variations less than 11.09%, whereas the mean accuracy ranged from 98.38% to 105.13%. The assay method exhibited good recovery and negligible matrix effect. The samples were stable under all the experimental conditions. The plasma concentrations of Eggmanone were detected and quantified over 24 h with the terminal elimination half-live of 3.57 ± 1.80 h and 5.92 ± 3.34 h for the low dose (1 mg/kg) and high dose (3 mg/kg) respectively. In summary, the present method provides a robust, fast and sensitive analytical approach for quantification of Eggmanone in plasma and was successfully applied to a pharmacokinetic study in rats.

Method Development and Validation for the Determination of Caffeine: An Alkaloid from Coffea arabica by High-performance Liquid Chromatography Method.

Objective:The present study was investigated to develop and validate a reversed phase high performance liquid chromatography method for the determination of caffeine from bean material of Coffee arabica.Materials and Methods:The separation was achieved on a reversed-phase C18 column using a mobile phase composed of water: methanol (50:50) at a flow rate of 1.0 mlmin-1. The detection was carried out on a UV detector at 272 nm. The developed method was validated according to the requirements for International Conference on Harmonisation (ICH) guidelines, which includes specificity, linearity, precision, accuracy, limit of detection and limit of quantitation.Results:The developed method validates good linearity with excellent correlation coefficient (R2 > 0.999). In repeatability and intermediate precision, the percentage relative standard deviation (% RSD) of peak area was less than 1% shows high precision of the method. The recovery rate for caffeine was within 98.78% - 101.28% indicates high accuracy of the method. The low limit of detection and limit of quantitation of caffeine enable the detection and quantitation of caffeine from C. arabica at low concentrations.Conclusion:The developed HPLC method is a simple, rapid, precisely, accurately and widely accepted and it is recommended for efficient assays in routine work.SUMMARYA simple, accurate, and sensitive high-performance liquid chromatography (HPLC) method for caffeine from Coffea arabica has been developed and validated. The developed HPLC method was validated for linearity, specificity, precision, recovery, limits of detection, and limits of quantification by the International Conference on Harmonization guidelines. The results revealed that the proposed method is highly reliable. This method could be successfully applied for routine quality work analysis.Abbreviation Used: C. arabica: Coffee arabica, ICH: International Conference on Harmonisation, % RSD: Percentage Relative Standard Deviation, R2: Correlation Coefficient, ppm: Parts per million, LOD: Limits of detection, LOQ: Limits of quantification, SD: Standard deviation, S: Slope, RP-HPLC: Reverse phase high performance liquid chromatography, v/v: Volume per volume.

Development of Rapid and Sensitive Reverse Phase High Performance Liquid Chromatography Method for Estimation of Ketorolac Tromethamine in Proniosomal Gel

Development of Rapid and Sensitive Reverse Phase High Performance Liquid Chromatography Method for Estimation of Ketorolac Tromethamine in Proniosomal Gel

Antimutagenic activity of compounds isolated from Ajuga bracteosa Wall ex. Benth against EMS induced mutagenicity in mice.

Ajuga bracteosa Wall ex. Benth. (Lamiaceae) has been reported to possess many biological activities including antibacterial, antifungal, antispasmodic and antioxidant activity but there is no report as such on its mutagenic and/or anti-mutagenic activity. The aim of the present study was to isolate compounds from the methanol extract of the aerial parts of Ajuga bracteosa and determine their anti-mutagenic activity against the mutagen, EMS in animal model mice. The study was undertaken in order to corroborate the traditional use of the plant Ajuga bracteosa. The compounds were isolated from the methanol extract of the aerial parts of Ajuga bracteosa using silica gel column chromatography. Structural elucidation of the isolated compounds was done using spectral data analysis and comparison with literature. High performance liquid chromatography (HPLC) was used for the qualitative and quantitative determination of the isolated compounds in the crude methanol extract. The isolated compounds and standard drug were evaluated in vivo for antimutagenic activity against EMS induced mutagenicity taking mice as model organism by micronucleus and chromosomal aberration tests. Four major compounds were identified as 1) 14, 15-dihydroajugapitin 2) β- Sitosterol 3) Stigmasterol and 4) 8-O-acetylharpagide. A quick and sensitive HPLC method was developed for qualitative and quantitative determination of three isolated marker compounds from Ajuga bracteosa. 14, 15-dihydroajugapitin reduced the micronuclei by 85.10%, followed by β- Sitosterol (72.3%) while as 8-O-acetylharpagide reduced the micronuclei by 46%. It is therefore evident from the present study that the plant contains rich source of anticancer and antimutagenic drugs.

Simultaneous determination of methotrexate and metoclopramide in physiological fluids using RP-HPLC with ultra-violet detection; application in evaluation of polymeric nanoparticles

ABSTRACTMethotrexate (MTX) is an anticancer drug while metoclopramide (MCP) is an antiemetic agent. Both the drugs are commonly coprescribed to avoid the emesis caused by anticancer drug. In this study, a novel, rapid, sensitive, and cost-effective reverse-phase high-performance liquid chromatography method was developed and validated for simultaneous determination of the methotrexate and metoclopramide in biological and pharmaceutical samples using sparfloxacin as internal standard. The analytes were separated on a Kromasil 100-5C18 RP (250 × 4.6 mm, 5 µm) column, methanol, and 0.05% trifloroacetic acid (36:64 v/v) as mobile phase with a flow rate of 1 mL/min, detection wavelength of 290 nm, and column oven temperature at 40°C. Both the analytes were extracted from physiological fluids (bovine aqueous humor, vitreous humor, and human plasma) using mixture of methanol and 10% perchloric acid (50:50 v/v). The method was linear over the concentration range of 0.025–1.0 µg/mL for methotrexate and 0.030–1.0 µg/mL for metoclopramide. The % recovery from human plasma was 98.57 and 96.74% for MTX and MCP, respectively, while from aqueous humor and vitreous humor was 95.84 and 98.51% for MTX.The developed method was applied for in vitro release of MTX from polymeric nanoparticles and can be applied for analysis of pharmaceutical and biological samples containing both the drugs.

Ionic liquid-based cloud-point extraction of quercetin for its sensitive HPLC–UV determination in juice samples

An ionic liquid-based cloud-point extraction (IL-CPE) method was developed for the extraction of quercetin in juice samples before its determination by high-performance liquid chromatography (HPLC). 1-Butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) was used as the ionic liquid. The cloud-point extraction parameters such as sample pH, extraction temperature, extraction time, amount of ionic liquid, extraction volume, and salt concentration were carefully studied and optimized for the achievement of maximum extraction recovery. Under the optimized conditions, i.e., 20 min heating at 40 °C, 100 μL IL volume, pH 2.0, and no salt addition, a mean recovery of 92.5% and an enrichment factor of 20 were obtained for quercetin. Relative standard deviation of the method was 3.76% for 6 replicates, and the calculated detection limit (3σ) of quercetin was 0.002 mg L−1. The method, coupled to HPLC was successfully applied to the sensitive determination of quercetin in apple and gapes juice samples with quantitative recoveries.

Therapeutic drug monitoring of ponatinib using a simple high-performance liquid chromatography method in Japanese patients

A simple and highly sensitive high-performance liquid chromatography (HPLC) method was developed for the quantification of ponatinib in human plasma. The developed HPLC method was validated based on International D.S. Food and Drug Administration guidelines. This technique utilized a solid-phase extraction step and required only 200μL plasma for a single analysis. The lower limit of quantification for ponatinib was 1.0ng/mL. Coefficients of variation and accuracies for intra- and interday assays were less than 10.8% and within 13.7%, respectively. The precision and accuracy of our HPLC assay was suitable for pharmacokinetic studies of ponatinib. On day 8 after beginning ponatinib therapy with an initial dose of 15mg, patients having a ponatinib C0 of less than 23ng/mL by HPLC may require a dose adjustment to 30mg to obtain a C0 of 23ng/mL of more. The median ponatinib C0 in 6 Japanese patients taking a 15mg daily dose was 24.6ng/mL, which was greater than the target concentration of 23ng/mL, and that of patients taking 30mg increased to a plasma concentration of 48.0ng/mL. This novel treatment strategy using the HPLC method developed herein may be useful for routine ponatinib therapy.

Simultaneous determination of tolterodine and its two metabolites, 5-hydroxymethyltolterodine and N-dealkyltolterodine in human plasma using LC–MS/MS and its application to a pharmacokinetic study

Tolterodine is a nonselective muscarinic antagonist that is indicated for the overactive urinary bladder and other urinary difficulties. We developed and validated a simple, rapid and sensitive high-performance liquid chromatography analytical method utilizing tandem mass spectrometry (LC-MS/MS) for the quantitation of tolterodine and its major metabolites, 5-hydroxymethyltolterodine (5-HMT) and N-dealkyltolterodine (NDT), in human plasma. After liquid-liquid extraction with methyl t-butyl ether, chromatographic separation of the three analytes was achieved using a reversed-phase Luna Phenyl-hexyl column (100×2.0mm, 3μm particles) with a mobile phase of 10mM ammonium formate buffer (pH 3.5)-methanol (10:90, v/v) and quantified by MS/MS detection in electrospray ionization (ESI) positive ion mode. The retention time of tolterodine, 5-HMT, NDT, and internal standard (IS) were 1.4, 1.24, 1.33, and 1.26min, respectively. The calibration curves were linear over a range of 0.025-10ng/ml for tolterodine and 5-HMT, and 0.05-10ng/ml for NDT. The lower limit of quantifications using 200μl of human plasma was 0.025ng/ml for tolterodine and 5-HMT, and 0.05ng/ml for NDT. The mean accuracy and precision for intra- and inter-run validation of tolterodine, 5-HMT, and NDT were all within acceptable limits. These results showed that a simple, rapid and sensitive LC-MS/MS method for the quantification of tolterodine and its major metabolites in human plasma was developed. This method was successfully applied to a pharmacokinetic study in humans.

HPLC method development and validation for the estimation of Axitinib in rabbit plasma

A rapid, sensitive, and accurate high performance liquid chromatography for the determination of axitinibe (AN) in rabbit plasma is developed using crizotinibe as an internal standard (IS). Axitinibe is a tyrosine kinase inhibitor, used in the treatment of advanced kidney cancer, which works by slowing or stopping the growth of cancer cells. The chromatographic separation was performed on a Waters 2695, Kromosil (150 mm × 4.6 mm, 5 µm) column using a mobile phase containing buffer (pH 4.6) and acetonitrile in the ratio of 65:35 v/v with a flow rate of1 mL/min. The analyte and internal standard were extracted using liquid-liquid extraction with acetonitrile. The elution was detected by photo diode array detector at 320 nm.The total chromatographic runtime is 10.0 min with a retention time for axitinibe and IS of 5.685, and 3.606 min, respectively. The method was validated over a dynamic linear range of 0.002-0.2µg/mL for axitinibe with a correlation coefficient of r2 0.999.

A selective and sensitive high performance liquid chromatography assay for the determination of cycloserine in human plasma

BackgroundCycloserine (CYC) is a second line antitubercular drug that is used for the treatment of multidrug resistant tuberculosis (MDR-TB) along with other antitubercular agents and is often used in developing countries. Monitoring CYC levels in plasma could be useful in the clinical management of patients with MDR-TB. A high performance liquid chromatography method for the determination of CYC in human plasma was developed. MethodsThe method involved extraction of the sample using solid phase extraction cartridges and analysis of the extracted sample using a reverse phase T3 column (150mm) and detection at 240nm with Photo Diode Array (PDA) detector. The chromatogram was run for 15min at a flow rate of 0.4ml/min at 30°C. Results and conclusionThe assay was specific for CYC and linear from 5.0 to 50.0μg/ml. The relative standard deviations of within- and between-day assays were less than 10%. Recovery of CYC ranged from 102% to 109%. The interference of other second line anti-TB drugs in the assay of CYC was ruled out. The assay spans the concentration range of clinical interest. The specificity and sensitivity of this assay makes it highly suitable for pharmacokinetic studies.

Development of Bioanalytical HPLC Method for Estimation of Milnacipran Hydrochloride in Rabbit Plasma Using Solid Phase Extraction Technique and its Application in Pharmacokinetic Investigation

Background: The availability of a low-cost bioanalytical method, easy to transfer and to set up, represents an advantage in therapeutic drug monitoring and industrial research. Objective: A simple, sensitive and rapid high performance liquid chromatographic (HPLC) method is developed and validated for quantitative determination of milnacipran hydrochloride in rabbit plasma. Method: Simple and effective solid phase extraction technique was performed for sample treatment and it has resulted in consistent and high recoveries (93–95%) at all concentrations studied. Efficient chromatographic separation has been performed on a LiChrospher® C18 column using a mobile phase consisting of a mixture of phosphate buffer, acetonitrile and methanol (65:25:10; v/v/v) at a flow rate of 0.8 mL/min. Results: The method has demonstrated linearity from 25 ng to 2000 ng/mL with a regression coefficient of 0.9998. The accuracy was found to be very high (99.31 to 101.44 %). %RSD values for inter-day and intra-day variation were not more than 3.58. The method has demonstrated high sensitivity with a lower limit of quantification of 25 ng/mL and excellent stability of milnacipran in rabbit plasma. The method was applied for pharmacokinetic investigation of immediate release and controlled release milnacipran tablets. The in-vivo study results indicated that developed HPLC method was sensitive to accurately quantify the concentration of milnacipran in plasma, which is an essential requirement for determination of pharmacokinetic parameters for drug formulation studies as well as future bioavailability or bioequivalence studies. Conclusion: A simple and sensitive bioanalytical HPLC method was developed, validated and applied for pharmacokinetic studies. Keywords: Milnacipran, liquid chromatography, solid phase extraction, rabbit plasma, pharmacokinetic investigation.

Design and Validation of Stability Indicating Assay of Glibenclamide Using RP-HPLC Technique in Both Bulk, Pharmaceutical Formulations and Human Plasma

A simple and sensitive high performance liquid chromatography method was developed for determination of glibenclamide in presence of its impurities in pharmaceutical dosage form and human plasma. Instrumentation of the method was very simple and used the mixture of acetonitrile: water (60:40, v/v) as the mobile phase. Separation was carried out on a BDS. Hypersil C8 (5 um, 250 × 4.6 mm) column. Effects of composition of mobile phase in addition to flow rate and detection wave length were studied. Calibration was obeyed in the range of 20-100 μg/ml of glibenclamide. The method was validated according to ICH parameters.

In-vitro and in-vivo pharmacokinetics of IS01957, p-coumaric acid derivative using a validated LC–ESI–MS/MS method in mice plasma

Plant derived natural products have been the major source for treatment of diseases traditionally but with the advent of modern systems of medicine, there is need to explore the active constituents present in it followed by modification for better therapeutic activity, low toxicity and favorable pharmacokinetics to become a drug molecule. A simple, rapid and sensitive high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated according to Food and Drug Administration guidelines for determination of IS01957, a derivative of naturally occurring para coumaric acid in mice plasma. The extraction of the analyte and the internal standard (Carbamazepine) from the plasma samples involved protein precipitation using acetonitrile. Results of validation parameters were met with the acceptance criteria of the FDA guidelines. Method was highly sensitive (5 ng/mL) that could determine very low concentration of compound in plasma The developed and validated method was successfully applied to determine compound’s metabolic stability in mouse liver microsomes (MLM) and human liver microsomes (HLM). Test compound was found to be stable in MLM and HLM in the experimental conditions. Metabolic stability data was extrapolated which was further correlated to pharmacokinetics study in mice through oral, intraperitoneal and intravenous administration. In-vitro half life was found to be greater than 2 h in both MLM and HLM. Hepatic extraction ratio of the compound was found to be in the intermediate range. Pharmacokinetic evaluations revealed that it is a suitable candidate for intraperitoneal as well as oral administration.

Quantification of theophylline or paracetamol in milk matrices by high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatography (HPLC) method was developed, validated and applied to the determination of either theophylline or paracetamol in milk-based samples. The method allowed drug quantification in fresh and powdered milk with a relatively short run time of analysis and it was also successfully applied to the quantification of the drugs in solid dosage forms intended for pediatric use. Moreover, the main significant advantages over other published works are the simplicity of the sample preparation, reduced assay time and sample loss. The method meets the International Conference on Harmonization guideline for analytical methods validation regarding specificity, linearity, accuracy, precision, specificity and robustness as required by health authorities and applied by industry while designing and marketing new drug products. The technique encompasses the separation of the analytes with a reverse phase C18 column under isocratic conditions and UV detection at 272nm and 243nm, respectively, for theophylline and paracetamol. The lower limit of quantification for both drugs was determined as 0.2µg/mL and the between-batch accuracy was approximately 99.7%. This HPLC method allows quantification of theophylline and paracetamol in milk matrices and it can be applied in the design, development and production of milk-based pediatric dosage forms.

Simultaneous and Sensitive Determination of Levodopa and Carbidopa in Pharmaceutical Formulation and Human Serum by High Performance Liquid Chromatography with On-Line Gold Nanoparticles-Catalyzed Luminol Chemiluminescence Detection

Abstract Levodopa, the metabolic precursor of dopamine, is usually administrated in combination with carbidopa to control dopamine levels in an appropriate manner and reduce side effects in the treatment of Parkinson's disease. In this study, a selective and sensitive high performance liquid chromatography coupled with on-line gold nanoparticles-catalyzed luminol chemiluminescence method for simultaneous determination of levodopa and carbidopa was developed. This method was based on the strongly enhanced chemiluminescence signal of on-line gold nanoparticles-catalyzed luminol-H2O2 system by levodopa and carbidopa. The possible enhancement mechanism was attributed to that levodopa and carbidopa could promote the on-line formation of a large number of gold nanoparticles, which catalyzed the luminol-H2O2 chemiluminescence reaction. The good separation of levodopa and carbidopa was achieved with isocratic elution using a mixture of methanol and 0.2% aqueous phosphoric acid (5:95, V/V) within 10.5 min. Under the optimal conditions, the linear ranges of levodopa and carbidopa were 2.24–448 ng mL−1 and 4.32–1080 ng mL− with the detection limits of 0.89 and 1.08 ng mL− (S/N = 3), corresponding to 17.92 and 21.60 pg for 20 μL sample injection, respectively. The validated method was successfully applied to simultaneous quantification of levodopa and carbidopa in controlled-release tablets (Sinemet®) and human plasma. The average recoveries of levodopa and carbidopa in the tablets were 100.5% and 103.1% with the precisions (RSDs) of 2.4% and 4.0%. The recoveries of levodopa and carbidopa in human plasma ranged from 97.0% to 103.5% with RSDs of no more than 3.3%.