Intra-and Inter-day Precision Research Articles

An Improved HPLC Method for Quantification of Metanephrine with Coulometric Detection

A rapid and straightforward analytical method, based on the use of RP-HPLC with coulometric detection, was developed and validated for the quantification of metanephrine, an O-methylated product in catechol-Omethyltransferase enzymatic assays. The isocratic separation was achieved on a reverse column with a mobile phase consisting of 0.1 M sodium dihydrogen phosphate, 0.024 M citric acid monohydrate, 0.5 mM sodium octyl sulphate and 9% acetonitrile (%v/v). The method was found to be linear between 0.25 and 15 nmol/mL with a determination coefficient of 0.9997 for metanephrine. Intra-and interday precision and accuracy were in conformity with the criteria accepted in bioanalytical method validation and the LOD and LLOQ were 0.25 nmol/mL. The main focus of the developed method is the lower LLOQ achieved that can have important implications in laboratory research for COMT activity determinations, in particular for the methionine 108/158 variant obtained either from native or recombinant extracts. Another major advantage of the present method is the shorter run times on automated chromatographic systems that allow the analysis of several samples in a short time. In addition, metanephrine was stable in the samples for at least 24 h at room temperature, for at least 24 h in HPLC system injector and for at least three freeze/thaw cycles. The developed method demonstrated higher sensitivity, precision, accuracy, stability, and linearity when compared with the methods previously described. Finally, a catechol-O-methyltransferase

A Novel Method for Determination of Fenofibric Acid in Human Plasma using HPLC-UV: Application to a Pharmacokinetic Study of New Formulations

A high performance liquid chromatography method for the determination of fenofibric acid (FA), the active form of fenofibrate (FBT) in human plasma was developed and validated with 500 μL of human plasma using 4’-chloro- 5-fluro-2-hydroxybenzophenone (CFHB) as internal standard (IS). The assay procedure involved a simple one step liquid/liquid extraction of FA and IS from human plasma into ethyl acetate. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40°C. The residue was reconstituted in the mobile phase and injected on a Symmetry ShieldRP18 (150×4.60 mm) 5 μm column. Separation of FA and IS was achieved with a mobile phase consisting of acetonitrile: 0.02 M phosphoric acid (50:50 v/v) at a flow rate of 1 mL/min. Nominal retention times of FA and IS were 6.1 and 9.1 ± 0.5 min, respectively. Absolute recovery of FA using a single step liquid/liquid method was 79.8%. A calibration curve was established for a range of concentrations 0.05 to 10.0 μg/mL with a regression coefficient (r2) of 0.9988. The lower limit of quantification (LLOQ) of FA was 0.05 μg/mL. The intraand inter-day precision for the measurement of FA quality control samples (0.05, 0.12, 1.20 and 8.20 μg/mL), were in the range 4.6-16.9% and 4.4-17.2% relative standard deviations respectively. The accuracy in the measurement of QC samples for FA was in the range of 82.0-104.3% (intra-day) to 95.0-104.9% (inter-day). The method developed was successfully used to investigate the pharmacokinetics and bioequivalence between a micronized Lipidil-Micro™ capsule formulation and a nanonised fenofibrate Lipidil-EZ™ tablet formulation.

Determination of a novel diacylglycerol acyltransferase 1 inhibitor, 2‐[4‐(4‐{5‐[2‐phenyl‐5‐(trifluoromethyl) oxazole‐4‐carboxamido]‐1H‐benzo[d]imidazol‐2‐yl} phenyl) cyclohexyl] acetic acid (KR‐69232) in rat plasma using liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

A liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of KR-69232, a diacyltransferase 1 inhibitor, in rat plasma. KR-69232 in the concentration range of 0.004-4 µg/mL was linear. The intra-and inter-day precision and accuracy were acceptable (<20%). KR-69232 was stable under various storage and handling conditions. The method was applied successfully in a pharmacokinetic study of KR-69232 in rats.

Simultaneous determination of phenolic acids by UPLC–MS/MS in rat plasma and its application in pharmacokinetic study after oral administration of Flos Lonicerae preparations

The current study aims to investigate the pharmacokinetic study of five phenolic acids (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 3,5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic acid) following oral administration of Flos Lonicerae preparations in rats. A rapid and sensitive ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed to simultaneously determine the five phenolic acids in rat plasma. After mixing with the internal standard (IS) tinidazole, plasma samples were pretreated by liquid–liquid extraction with ethyl acetate/n-hexane (9:1, v/v). The separation was performed on an Acquity UPLC BEH C18 column (100mm×2.1mm, 1.7μm) at a flow rate of 0.4mlmin−1, and acetonitrile/methanol (4:1, v/v)-0.4% formic acid was used as mobile phase. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) via electrospray ionization (ESI) source with positive ionization mode. All calibration curves had good linearity (r>0.991) over the concentration ranges of 0.74–378ngml−1 for neochlorogenic acid, 0.50–1030ngml−1 for chlorogenic acid, 1.9–250ngml−1 for cryptochlorogenic acid, 0.74–380ngml−1 for 3,5-dicaffeoylquinic acid, and 5.1–328ngml−1 for 3,4-dicaffeoylquinic acid. The intra-and inter-day precision were within 15% and the accuracy ranged from 86.2% to 114.1%.

Preparation and Evaluation of Histamine Imprinted Polymer as a Selective Sorbent in Molecularly Imprinted Solid-Phase Extraction Coupled with High Performance Liquid Chromatography Analysis in Canned Fish

A series of molecularly imprinted polymers (MIPs) were prepared against histamine. Different template/monomer ratios were applied to optimize the imprinting condition. Methacrylic acid (MAA) as a functional monomer and Chloroform as a solvent were applied in polymerization process. The binding properties of MIPs were studied in comparison with a blank non-imprinted polymer. The optimized polymer, with a histamine/MAA ratio of 1/4, was selected as a sorbent in molecularly imprinted solid-phase extraction (MISPE) of histamine from canned fish. Scatchard analysis of MIP-histamine interactions revealed two types of binding sites for MIP: high affinity (KD = 11.11 μM) and low affinity (KD = 333.3 μM). The MISPE procedure was calibrated and a recovery of 76.5–97.6 % was obtained. The intra-and inter-day precision values were less than 5.70 % and 10.1 %, respectively. The selectivity of MISPE for histamine was also studied in comparison with some other structurally similar amines, which could be simultaneously present in canned fish. The performance of the imprinted polymer was examined and the results indicated that its good selectivity and affinity for histamine was very promising. Therefore, the proposed calibrated method could be applied in selective extraction and analysis of histamine in canned fish.

LC-MS/MS를 이용한 인체 혈장에서 Ginsenoside Rb1의 분석법 검증

A new liquid chromatographic tandem mass spectrometric (LC-MS/MS) assay for the quantification of ginsenoside Rb1 in human plasma was developed and validated. The separation was performed on a Agilent C18 column (4.6 mm×150 mm, particle size 5 μm) with a gradient elution of 0.1% formic acid in water and 0.1% formic acid in methanol and a flow rate of 0.9 mL/min. The analyte was determined using electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode (m/z 1131.714→365.303). Human plasma samples were extracted with acetone : water (50:50) by the liquid-liquid extraction method. The method was linear over the dynamic range of 10～500 ng/mL with a correlation coefficient of r=0.9995. The intra-and inter-day precision over the concentration range of ginsenoside Rb1 was lower than 5.8% (correlation of variance, CV), and the accuracy was between 96.0～104.6%. This LC-MS/MS assay of ginsenoside Rb1 in human plasma is applicable for quantification in a pharmacokinetic study.

Determination of the lincomycin hydrochloride content in lincomycin hydrochloride thermosensitive eye gel by RP-HPLC method

Objective:To establish a RP-HPLC method for determination of the lincomycin hydrochloride content in lincomycin hydrochloride thermosensitive eye gel.Methods:The separation was performed on a Diamond C18 column(250 mm×4.6 mm,5 μm) with the mobile phase consisting of 0.05 mol/L borax water solution(pH value adjusted to 7.4 by phosphoric acid)-methanol(50∶50) at the flow rate of 1.0 ml/min.The detection wavelength was 214 nm.Results:Lincomycin hydrochloride presented a good linearity within the range of 2-48 μg/ml(r=0.999 9).It had good intra-and inter-day precision,stability and reproducibility.The average recovery rates of low,middle and high concentrations(11.608,17.584,41.501 μg/ml) were(91.40±1.27)%,(97.55±0.93)% and(99.13±1.11)%(n=3),respectively.The average contents of lincomycin hydrochloride in three batches of samples were(98.67±1.21)%,(99.90±0.44)% and(99.28±1.17)%(n=3),respectively.Conclusion:It is an accurate,sensitive and reproducible method for determination of the lincomycin hydrochloride content in lincomycin hydrochloride thermosensitive eye gel.

Development and Validation of a GC-MS Method for Rapid Determination of Calophyllolide in Calophyllum inophyllum L.: A Quality Control Approach

Aim To establish a GC-MS method for the determination of calophyllolide in Calophyllum inophyllum L. and its application in quality control of natural medicine. Methods Methanolic extract from Calophyllum inophyllum L. was prepared. Calophyllolide was isolated, purified and then identified to serve as a chemical marker for development and validation a GC-MS method according to ICH guideline and USP. The established method was proved to be useful in quality control with the aid of Principle Component Analysis (PCA), a multivariate data analysis technique. Results The proposed method was selective against mass spectral deconvoluting software AMDIS32. The linear range was from 3.125–50 μg·mL −1. The RSD of intra-and inter-day precision variations were less than 3% and the mean recovery was (101.22 ± 1.98) % (RSD = 1.95%). PCA was used to indicate a differentiation of batches in production as well as proving that calophyllolide has influence on the discrimination of extracts from different batches. Conclusion The proposed capillary gas chromatography/mass spectrometric method for the determination of calophyllolide obtained from resin of Calophyllum inophyllum L. nut oil showed its suitability, simplicity, rapidity and precision, and it can be used for chemical assay. This is the first time such a technique was reported in literature. The multivariate data analysis technique was employed to provide an extensive but inclusive method for quality control of the extracts, to ensure consistency for batch to batch of extracts to be used for product development.

Validation and Applications of Gas Chromatography-Combustion/isotope Ratio Mass Spectrometric Method to Control Misuse of Androgens in Human

The misuse of anabolic androgenic steroids is of particular concern in sports and society. Thus, it is of great importance to discriminate endogenous steroids such as testosterone or testosterone prohormones from their chemically identical synthetic copies. In this study, gas chromatography-combustion/isotope ratio mass spectrometric (GC−C/IRMS) method has been developed and validated for discriminating the origin of anabolic androgenic steroids. The method involves the solid-phase extraction, enzymatic hydrolysis with β-glucuronidase, HPLC-fractionation for the cleanup and analysis by GC−C/IRMS. The difference (∆13C) of urinary δ13C values between synthetic analogues and endogenous reference compounds (ERC) by GC−C/IRMS was used to elucidate the origin of steroids, and intraand inter-day precision, specificity and isotope fractionation were evaluated. The present GC−C/IRMS method combined with HPLC cleanup was accurate and reproducible enough to be successfully applied to the test of urine sample from suspected anabolic steroid abusers.

High-performance liquid chromatographic determination and pharmacokinetic study of apigenin-7-O-β-D-glucoside in rat plasma after intravenous administration

The present study was designed to validate an analytical method based on HPLC for the quantitative measurement of the Apigenin-7-O-β-D-glucoside (AGL) in rat plasma after intravenous administration. HPLC analysis was done using a C18 column, UV detection at 335 nm, and a mobile phase of methanol:0.2% phosphoric acid (1:1, v/v). Good linearity was observed over the range of 0.06-7.20 μg/mL with a lower limit of quantication of 0.06 μg/mL. The intra-and inter-day precision values were below 9.97%. No interference peaks or matrix effects were observed. The method was fully validated and successfully applied to a pharmacokinetic study of AGL.

GC/MS를 이용한 요 중 resveratrol과 그 대사체에 관한 연구

This study was conducted to define metabolite of the resveratrol by gas chromatography- time-offlight mass spectrometric detection. From these results, we suppose that the structure of metabolite is the result of reduction of double-bond attached by two-phenyl groups. Also, validity of method for determining metabolite of resveratrol and endogenous steroids was tested. The recoveries ranged from 96.47 to 114.74%, and intraand inter-day precision ranged 11.40 - 10.87% and 1.10 - 10.93%, accuracy ranged 80.03 - 119.92% and 80.02 - 119.56%, respectively. Resveratrol and endogenous steroids had correlation coefficients above or equal to 0.996. The method was successfully validated for the determination of resveratrol and endogenous steroids. Urinary samples from volunteers dosed resveratrol were analyzed to confirm a correlation resveratrol and its metabolite. From these results, the highest level of resveratrol and its metabolite was excreted in 10 - 15 hr more slowly than common drug, and conversion rate of metabolite was higher in woman than that in man. In addition, endogenous steroids were shown same the highest level of 10 - 15 hr. For estrone and estradiol, sensitivity was relatively higher in female than in man. And there were no significant changes of excretion patterns in the other endogenous steroids. Thus, we assumed that activation of resveratrol has impact on woman than man.

Determination of Tigecycline in Human Skin Using a Novel Validated LC–MS/MS Method

To develop and validate a sensitive and novel bioanalytical method for measuring tigecycline concentrations in human skin using LC-MS/MS. The method utilizes addition of a stabilizing agent to the human skin or surrogate (human liver or rat skin), homogenization of human skin in a strong acidic-methanol extraction solvent, centrifugation of the skin suspension, filtration of the skin suspension supernatant, separation by LC (Polaris™ C18-A 50 × 2.0 mm), and detection of tigecycline by MS/MS. Linearity was 50-20,000 ng/g, using a sample size of 100 mg. The intra-and inter-day accuracy and precision of the assay met acceptance criteria. This method has been successfully applied to 17 incurred human skin samples from volunteers with surgical infections who received intravenous doses of tigecycline (100 mg initial loading dose and 50 mg every 12 h for at least 2 days). Tigecycline concentrations in these samples ranged from 185 to 2853 ng/g.

UPLC–MS–MS Analysis of Baicalin in the Cerebrospinal Fluid of Rabbits: Application to a Pharmacokinetic Study

A sensitive and selective ultra-performance liquid chromatographic–tandem mass spectrometric method has been developed for analysis of baicalin in the cerebrospinal fluid of rabbits. Samples were separated on a C18 column with a gradient prepared from acetonitrile and 0.3% aqueous formic acid solution as mobile phase. The target compounds were quantified by multiple reaction monitoring (MRM) using electrospray ionization (ESI). Intra-and inter-day accuracy, precision, and linear range were investigated in detail. The lower limit quantification (LOQ) was 0.108 ng mL−1. The method has been successfully used for evaluation of the pharmacokinetics of baicalin in 12 rabbits.

Determination of macelignan in rat plasma by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic method was developed for the determination of macelignan in rat plasma and applied to the pharmacokinetic study of macelignan in rats. Chromatographic separation was achieved on a conventional ODS column with the mobile phase of water: acetonitrile: methanol = 35:32.5:32.5 (v/v/v %). The flow rate of isocratic elution was 1 mL/min and peaks were detected at 240 nm. The limit of detection was 10 ng/mL and the limit of quantitation was 20 ng/mL. The calibration curve was linear over the concentration range of 50-5000 ng/mL. Intra-and inter-day precision for the assay over the concentration range was below 10 % and the accuracy ranged between 96.0-107% for intra-day and 98.8-114% for inter-day, respectively. The method was applied to the single dose pharmacokinetic study of macelignan in rats and the results showed that this HPLC method was adequate to support the in vivo pharmacokinetic study of macelignan.

Extraction and analysis of ellagic acid from novel complex sources

AbstractEllagic acid (EA) was quantified by reversed-phase high-performance liquid chromatography (RPHPLC) coupled with photodiode array detection (DAD) in five fine-powdered plants collected from the semiarid region of Mexico. Samples analysed included Jatropha dioica branches (Dragon’s blood), Euphorbia antisyphyllitica branches (Candelilla), Turnera diffusa Willd leaves (Damiana), Flourensia cernua leaves (hojasén) and Punica granatum husk (pomegranate) at two maturity stages (“turning” or intermediate and maturated fruit, considered as positive controls). The results demonstrated high EA concentrations in all tested samples which are novel sources of this natural antioxidant. The method developed for the EA analysis is fast and it showed an excellent linearity range, repeatability, intra-and inter-day precision and accuracy with respect to the methods reported for the EA analysis.

Determination of pitavastatin in human plasma via HPLC-ESI-MS/MS and subsequent application to a clinical study in healthy Chinese volunteers

Background Pitavastatin is a novel statin used in the treatment of hyperlipemia. We developed and describe a simple and rapid high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) assay for the determination of pitavastatin in human plasma. Methods A Finnigan TSQ Quantum Discovery max system equipped with an electrospray ionization source and a Finnigan Surveyor™ HPLC system (Thermo Electron, San Jose, CA) was used employing lovastatin as internal standard (IS) for pitavastatin. This method entailed a single step of liquid–liquid extraction with ether from 200 μL plasma. The analyte and internal standard were baseline separated on a Gemini analytical column. Quantitation by SRM analysis was performed in the positive ion mode. Results HPLC-ESI-MS/MS method validation by means of determination of limit of detection (LOD 0.05 ng/ml), lower limit of quantification (LLOQ 0.1 ng/ml), linearity (0.2–200 ng/ml). The intra-and inter-day precision CVs was < 10%, and accuracy ranged from 85 to 115%. The proposed method enables the unambiguous identification and quantification of pitavastatin for clinical studies. Conclusion A sensitive and specific method for quantifying Pitavastatin levels in human plasma has been devised and successfully applied to a clinic pharmacokinetic study of pitavastatin administered.

Rapid titrimetric and spectrophotometric assay methods for the determination of lamivudine in pharmaceuticals using iodate and two dyes

One titrimetric and two spectrophotometric methods, which are simple, sensitive and rapid, are described for the assay of lamivudine in bulk drug and in tablet dosage forms using potassium iodate and two dyes, methyl orange and indigocarmine, as reagents. In titrimetry, an aqueous solution of lamivudine is titrated directly with iodate in an acidic medium, and in the presence of an excess of bromide using methyl orange as an indicator. After the decoloration of the red color of methyl orange, the residual bromine is titrated iodometrically to a starch endpoint. Spectrophotometric methods involve the addition of a known excess of iodate in an acidic medium and in the presence of an excess of bromide followed by the determination of residual bromine by the reaction with a fixed amount of either methyl orange and measuring the absorbance at 520 nm (method A), or indigo carmine and measuring the absorbance at 610 nm (method B). In all methods, the amount of iodate which reacted corresponds to the amount of lamivudine content. The titrimetric method is applicable over the 1.5–8.0 mg range. The systems obey Beer’s law for 0.5–5.0 µg/mL (method A) and 1.25–12.5 μg/mL (method B). The calculated apparent molar absorptivity values are found to be 3.3 × 104 and 9.3 × 103 L mol−1 cm−1, for method A and method B, respectively, and the corresponding Sandell sensitivity values are 6.94 and 24.62 ng/cm2. The limits of detection and quantification are also reported for both spectrophotometric methods. Intra-and interday precision and accuracy for the developed methods have been evaluated. The methods were successfully applied to the assay of lamivudine in tablet form and the results were compared with those of a reference method by applying the Student’s t-test and F-test. No interference was observed from common tablet adjuvants. The accuracy and reliability of the methods were further ascertained by recovery experiments using the standard addition technique.

Development and optimization of an analytical method for the determination of Sudan dyes in hot chilli pepper by high-performance liquid chromatography with on-line electrogenerated BrO −-luminol chemiluminescence detection

The determination of four Sudan dyes by means of high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection was proposed. The method was based on the enhancement effect of Sudan dyes on the chemiluminescence reaction between luminol and BrO −, which was on-line electrogenerated by constant current electrolysis. The separation was carried out on Nucleosil RP-C 18 column (250 mm × 4.6 mm i.d., 5 μm, pore size, 100 Å) at 35 °C. The mobile phase consisted of a V (methanol): V (0.2% aqueous formic acid) = 90:10 solution. At a flow-rate of 1.0 ml min −1, the total run time was 25 min. The effects of several parameters on the HPLC resolution and CL emission were studied systematically. For the four Sudan dyes, the limits of detection (LOD) at a signal-to-noise of 3 ranged from 4 to 8 μg kg −1 and the limits of quantification (LOQ) at a signal-to-noise of 10 ranged from 13 to 27 μg kg −1. The relative standard deviations (RSD) of intra-and inter-day precision were below 4.4%. The average recoveries for all four Sudan dyes (spiked at the levels of 1.0 and 1.5 mg kg −1) in chilli tomato sauce and hot chilli pepper ranged from 94% to 105%, and the relative standard deviations of the quantitative results were from 2.5 to 4.2%. The proposed method had been successfully applied to the determination of four Sudan dyes in hot chilli products.

Simultaneous LC Determination of Major Constituents in Red and White Peony Root

A reversed phase high performance liquid chromatography method was established for the simultaneous determination of eight major constituents, namely gallic acid, paeoniflorin sulfonate, catechin, albiflorin, paeoniflorin, benzoic acid, pentagalloylglucose and benzoylpaeoniflorin in red and white peony roots, the two commonly used traditional Chinese medicinal herbs. The optimal conditions of separation and detection were achieved on a C18 analytical column with a gradient mobile phase consisting of acetonitrile and 0.015% phosphoric acid at the flow rate of 1.0 mL min−1 and detection wavelength set at 230 nm. All calibration curves showed good linear regression (r>0.9995) within test ranges. This method provided good reproducibility with overall intra-and inter-day precision of less than 5% and 4% and good accuracy with recovery of more than 93%, respectively. The method was successfully applied to determine 71 samples of red and white peony roots collected from different areas. The results indicated that the contents of eight compounds varied significantly among the samples determined, which mainly resulted from processing procedure and habitat variation. The roots of Paeonia veitchii Lynch. contained a much higher amount of gallic acid and pentagalloylglucose than that of Paeonia lactiflora Pall., which could be used to distinguish the two similar species.

Determination of Omeprazole in Bulk and Injectable Preparations by Liquid Chromatography

An accurate, simple, reproducible, and sensitive liquid chromatographic method was developed and validated for the determination of omeprazole in powder for injection and in pellets. The analyses were performed at room temperature on a reversed-phase C18 column of 250 x 4.6 mm id, 5 microm particle size. The mobile phase, composed of methanol-water (90 + 10, v/v), was pumped at a constant flow rate of 1.5 mL/min. Detection was performed on a UV detector at 301 nm. The method was validated in terms of linearity, precision, accuracy, and ruggedness. The response was linear in the range 32-48 microg/mL (r2 = 0.9976). The relative standard deviation values for intra- and interday precision studies were 1.22 and 1.56% for injectable and 2.13 and 2.45% for pellets, respectively. Recoveries ranged between 95.81 and 100.48%.