Development and validation of a UPLC‐UV method for the determination of daptomycin in rabbit plasma

Daptomycin (DPT) is a lipopeptide antibiotic with potent bactericidal activity in vitro against Gram‐positive bacteria, which has attracted the attention of the scientific community due to its unique mechanism of action and due to the immediate need for new antibiotics in the era of multidrug resistance. In order to assess its pharmacokinetics in rabbits a new analytical method has been developed and validated using ultra performance liquid chromatography in conjugation with ultraviolet detection for the quantitation of the antibiotic in rabbit plasma, using the internal standard methodology. The separation was achieved employing a C₁₈ column with gradient elution using 0.1% aq. trifluoroacetic acid and methanol. The total analysis time was 2.5 min. The sample pretreatment employed protein precipitation with acetonitrile–methanol mixture and centrifugation. The method was validated in terms of linearity, precision, accuracy, sensitivity, robustness, short‐term and freeze–thaw stability and was applied to the quantification of DPT in plasma samples obtained from rabbits treated with 25 mg kg^?1 DPT. Copyright © 2009 John Wiley & Sons, Ltd.     

High-performance liquid chromatographic method for the determination of mangiferin in rat plasma and urine

A reversed-phase high-performance liquid chromatography assay for mangiferin in rat plasma and urine was developed. Rutin was employed as an internal standard. The mobile phase consisted of acetonitrile-water (16:84, v/v) containing 3% acetic acid at a flow rate of 1 mL/min. Detection was at 257 and 365 nm for mangiferin in plasma and urine, respectively. The limit of quantitation (LOQ) of mangiferin was 0.6 microg/mL in plasma, and 0.48 microg/mL in urine. The standard curve was linear from 0.6 to 24 microg/mL in plasma, and 0.48 to 24 microg/mL in urine, both intra- and inter-day precision of the mangiferin were determined and their RSD did not exceed 10%. The method provides a technique for rapid analysis of mangiferin in rat plasma and urine, which can be used in pharmacokinetic studies.     

An LC-MS-MS method for quantitative determination of maraviroc (UK-427,857) in human plasma, urine and cerebrospinal fluid

Maraviroc is a first‐in‐class CCR5 antagonist that shows potent anti‐HIV‐1 activity in vitro and in vivo and is well tolerated in both healthy volunteers and HIV‐1‐infected patients. The method for determination of maraviroc (UK‐427,857) and its major metabolite (UK‐408,027) in human plasma consists of a protein‐precipitation procedure and analysis by liquid chromatography/tandem mass spectrometry using positive ion TurboIonSpray® ionization and multiple reaction monitoring. The assay has been validated over a concentration range of 0.500–500 ng/mL for both analytes. The determinations of maraviroc in human cerebrospinal fluid (0.500–500 ng/mL) and in urine (5.00–5000 ng/mL) have also been validated but do not include measurement of the metabolite. The validations included extraction recovery, intra‐assay and inter‐assay precision and accuracy, stability of stock and spiking solutions, freeze–thaw stability, matrix stability, processed‐extract stability, and evaluation of potential interferences from selected medications in plasma or urine. Copyright © 2010 John Wiley & Sons, Ltd.     

Simple method for the assay of clindamycin in human plasma by reversed-phase high-performance liquid chromatography with UV detector

A rapid and simple high-performance liquid chromatography (HPLC) method was developed and validated for the quantification of clindamycin in human plasma. After precipitation with 50% trichloroacetic acid (TCA) containing the internal standard, propranolol, the analysis of the clindamycin level in the plasma samples was carried out using a reverse-phase cyano (CN) column with ultraviolet detection (204 nm). The chromatographic separation was accomplished with an isocratic mobile phase consisting of acetonitrile-distilled water-7.6 mm tetramethylammonium chloride (TMA) (60:40:0.075, v/v/v), adjusted to pH 3.2. The proposed method was specific and sensitive with a lower limit of quantitation (LLOQ) of 0.2 microg/mL. This HPLC method was validated by examining the precision and accuracy for inter- and intraday analysis in the concentration range 0.2-20.0 microg/mL. The relative standard deviations (RSD) in the inter- and intraday validation were 6.1-14.9 and 6.0-16.1%, respectively. In the stability test, clindamycin was found to be stable in human plasma during the storage and assay procedure. The present HPLC method was applied to the analysis of samples taken up to 12 h after a single oral administration of clindamycin in healthy volunteers.     

Quantitative determination of uridine in rabbit plasma and urine by liquid chromatography coupled to a tandem mass spectrometry

Recently a pyrimidine nucleoside, uridine, has been show to have a protective effect on cultured human corneal epithelial cells, and on dry eye animal model and patients. In this study, we introduce a sensitive liquid chromatography/tandem mass spectrometry method for the determination of uridine in rabbit plasma and urine. After protein precipitation with methanol including methaqualone (internal standard), the analyte was chromatographed on a reversed-phase column with a mobile phase of 0.1% formic acid aqueous solution and methanol (1:4, v/v). The accuracy and precision of the assay were in accordance with Food and Drug Administration regulations for the validation of bioanalytical methods. This method was used to measure the concentrations of uridine in plasma and urine after a single oral administration of 450 mg/kg uridine in rabbits.     

Development of a method for the determination of vardenafil in human plasma by high performance liquid chromatography with UV detection

A simple high‐performance liquid chromatographic (HPLC) method with photometric detection is described for the determination of vardenafil hydrochloride, a phosphodiesterase V inhibitor, in human plasma. Chromatographic separation of the analyte and internal standard was achieved on an analytical 250 × 4.6 mm i.d. reversed‐phase Kromasil KR 100 C₁₈ (5 µm particle size) column using a mobile phase of acetonitrile–potassium dihydrogen phosphate (30:70 v/v). The run time was less than 15 min. Column eluate was monitored at 230 nm. The linearity over the concentration range of 10–1500 ng/mL for vardenafil was obtained and the limit of quantification (LOQ) was 10 ng/mL. The method has been applied to analysis of the vardenafil concentrations for application in pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Meropenem and ciprofloxacin in complicated gastric surgery for cancer patients: A simple SPE–UHPLC–PDA method for their determination in human plasma

A simple and rapid ultra‐high‐performance liquid chromatographic (UHPLC) for the simultaneous determination of meropenem and ciprofloxacin in human plasma was developed and validated. All of the analytes were separated in <5 min. A solid‐phase extraction method was applied from sample preparation. Analytical separation was performed on a Poroshell SB C₁₈ column (50 × 2.1 mm, 2.7 μm particle size) with photodiode array (PDA) detection. Meropenem and ciprofloxacin were determined at wavelengths of 300 and 277 nm, respectively. The mobile phase was a mixture of acetonitrile–10 mm ammonium acetate–methanol in gradient elution. The method has been validated for both drugs in gastric surgery for cancer patients. The method showed good linearity with correlation coefficients, r² = 0.994 for the two drugs, as well as high precision (RSD < 10.5% in each case); accuracy ranged from ?5.8 to +6.0%. The limit of quantitation of the two drugs was established at 0.02 and 0.01 μg/mL, respectively. Meropenem, ciprofloxacin and the internal standard were extracted from human plasma with a mean recovery ranging from 92.5 to 98.6%. The method was applied to quantify the drugs dosage in complicated gastric surgery patients.     

Simple determination of huperzine A in human plasma by liquid chromatographic-tandem mass spectrometric method

Huperzine A is a potent, reversible acetylcholinesterase inhibitor. In the present work, a rapid and sensitive LC-MS-MS method for the determination of huperzine A in human plasma using codeine phosphate as internal standard has been developed and validated. The analyte and internal standard were extracted from plasma using ethyl acetate, chromatographed on a C(18) column (5 microm, 150 x 4.6 mm i.d.) with a mobile phase consisting of 1% formic acid-methanol (40:60, v/v), and detected using a tandem mass spectrometer with a TurboIonSpray ionization interface. The run time was only 2 min. Good linearity was achieved in the range 0.126 -25.2 ng/mL and the limit of detection in plasma was 0.064 ng/mL. The average recovery for huperzine A was 83.4% from plasma. The analytical sensitivity and accuracy of this assay is adequate for characterization of huperzine A in human plasma.     

Accurate,sensitive determination of omega-6 and omega-3 polyunsaturated fatty acids in human plasma,urine samples

Quantitative determination of omega-6 and omega-3 polyunsaturated fatty acids in human plasma and urine with high accuracy and precision provides significant information to monitor the underlying etiology of several diseases. In this regard, liquid chromatography-mass spectrometry is a good choice owing to its great selectivity and sensitivity. Additionally, the hybrid quadrupole–time of flight–mass spectrometer systems provides easy identification of target compounds with superior mass measurements. In this study, an analytical method has been developed for simple, accurate and simultaneous determination of linoleic acid, arachidonic acid, docosahexaenoic acid and eicosapentaenoic acid in a short chromatographic analysis period. The developed method is suitable for the quantitative detection of these four compounds with detection limits ranging between 1.1–3.0 ng ml⁻¹ and its applicability was assessed in human urine and plasma samples. As a result, acceptable accuracy (between 83 and 111%) and good precision (<6%) were obtained for target compounds using matrix matching calibration strategy.     

Simple and rapid determination of piperacillin and ceftazidime in human plasma samples by HPLC

Summary A simple and rapid liquid chromatographic method has been developed for the determination of therapeutic levels of piperacillin (I) and ceftazidime (II) in human plasma. Plasma and p-propionamidophenol (internal standard) were precipitated with methanol (I) or 20% trichloroacetic acid (II). The supernatant was analysed on a 5 μm Spherisorb ODS C₁₈ column with acetonitrile-0.05 M phosphate buffer pH 3.8 as mobile phase and ultraviolet detection at 254 nm. The calibration graph was linear from 10 to 250 μg mL⁻¹, for (I), and from 5 to 200 μg mL⁻¹ for (II). Intra and inter-day CV did no exceed 2.29% for (I), and were 10.76–11.13%–2.00–5.62 for (II) at concentrations of 10 μg mL⁻¹ and 250 μg mL⁻¹.     

High performance liquid chromatographic determination of metoclopramide in plasma and urine and its application to biopharmaceutical investigations

An optimized HPLC method for the quantification of metoclopramide (MCP) in human plasma and urine is described. MCP and internal standard are extracted from alkalinized substrate into diethyl ether and back-extracted into dilute acid. The analytes are separated with a ternary mobile phase at cyanopropyl-silica and detected at 312 nm (UV detection). The lower limit of quantification is 0.5 ng/ml in plasma and 50 ng/ml in urine. Optimization of extraction, chromatography, and detection is discussed. The method is selective to numerous common drug substances with excellent accuracy and precision data. After validation, the method is applied to the samples of a pharmacokinetic study. Pharmacokinetic parameters indicate the need for a sophisticated method as tool for optimization of metoclopramide formulations.     

High-throughput liquid-chromatography method with fluorescence detection for reciprocal determination of furosemide or norfloxacin in human plasma

A simple, high-throughput, highly selective and sensitive HPLC-FLD method for isolation and determination of furosemide and/or norfloxacin in human plasma samples following a simple organic solvent deproteinization step with acetonitrile as sample 'clean-up' procedure is reported. One of the two drug substances plays the internal standard role for the determination of the other. Separation of analyte and internal standard was achieved in less than 5.3 min (injection to injection) on a Chromolith Performance RP-18e column, using an aqueous component containing 0.015 mol/L sodium heptane-sulfonate and 0.2% triethylamine brought to pH = 2.5 with H(3)PO(4). The composition of the mobile phase was: acetonitrile-methanol-aqueous component = 70:15:15 (v/v/v) and the flow-rate was set up to 3 mL/min. The chromatographic method applied to the determination of furosemide relies on fluorescent detection parameters of 235 nm for the excitation wavelength, and 402 nm for the emission wavelength. In case of norfloxacin, the excitation wavelength is set up to 268 nm and the emission wavelength is set up to 445 nm. The overall method leads to quantitation limits of about 27 ng/mL for furosemide, and 19.5 ng/mL for norfloxacin, using an injection volume of 250 microL. The method was applied to the bioequivalence study of two furosemide-containing formulations.     

A liquid chromatography-tandem mass spectrometry assay for the determination of nemonoxacin (TG-873870), a novel nonfluorinated quinolone, in human plasma and urine and its application to a single-dose pharmacokinetic study in healthy Chinese volunteers

Nemonoxacin (TG‐873870) is a novel C‐8‐methoxy nonfluorinated quinolone with higher activity than ciprofloxacin, levofloxacin and moxifloxacin against Gram‐positive pathogens including methicillin‐susceptible or methicillin‐resistant Staphylococcus aureus and Streptococcus pneumoniae with various resistant phenotypes. A rapid, sensitive and selective liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method was developed and validated to determine the concentration of nemonoxacin in human plasma and urine. Protein precipitation and liquid–liquid extraction were employed for plasma and urine sample preparations, respectively, and extract was then injected into the system. Separation was performed on a C₁₈ reversed‐phase column using acetonitrile–0.1% formic acid as a mobile phase. Both analyte and internal standard (gatifloxacin) were determined using electrospray ionization and the MS data acquisition via the selected reaction monitoring in positive‐ion mode. The lower limit of quantification was 5 ng/mL and the calibration curves were linear in the concentration range of 5–1000 ng/mL. The accuracy, precision, selectivity, linearity, recovery, matrix effect and stability were validated for TG‐873870 in human plasma and urine. The method was successfully applied to a pharmacokinetic study enrolling 12 healthy Chinese volunteers administered nemonoxacin malate capsules. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of HPLC-MS/MS method for the simultaneous determination of environmental phenols in human urine

We present a sensitive method for simultaneous determination of bisphenol A (BPA), benzophenone-3 (BP-3), 4-tert-octylphenol (t-OP), ortho-phenylphenol (OPP), four parabens (methyl, ethyl, propyl, butyl parabens) and five chlorophenols (2,4-dichlorophenol (2,4-DCP), 2,5-dichlorophenol (2,5-DCP), 2,4,5-trichlorophenol (2,4,5-triclorophenol), 2,4,6-trichlorophenol (2,4,6-TCP), and triclosan (TCS)), in human urine by high-pressure liquid chromatography (HPLC) mass spectrometry (MS). Samples were processed using enzymatic deconjugation of glucuronides followed by solid phase extraction (SPE) on a C18 cartridge and the eluate was concentrated. Analytes were separated by reversed-phase HPLC and then detected by atmospheric pressure chemical ionisation (APCI) MS and quantified by isotope dilution method. We describe details for optimisation of each step of the procedure. The sample treatment steps are straightforward and not labour-intensive and, therefore, permit a high sample throughput with excellent prospects for automation. This method shows low inter-day variation, and detection limits for most of the compounds are below 1 ng/mL in 1 mL of urine. The method accuracy was also verified by the analysis of proficiency testing urine samples.     

Simultaneous determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using ultra-performance liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

A rapid, sensitive, and simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method for the determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using sildenafil as an internal standard (IS) was developed and validated. Udenafil, DA-8164 and IS from a 100 microL aliquot of biological samples were extracted by protein precipitation using acetonitrile. Chromatographic separation was carried on an Acquity UPLC BEH C(18) column (50 x 2.1 mm, i.d., 1.7 microm) with an isocratic mobile phase consisting of acetonitrile and containing 0.1% formic acid (75:25, v/v) at flow rate of 0.4 mL/min, and total run time was within 1 min. Detection and quantification was performed by the mass spectrometer using multiple reaction-monitoring mode at m/z 517 --> 283 for udenafil, m/z 406 --> 364 for DA-8164 and m/z 475 --> 100 for IS. The assay was linear over a concentration range of 1-600 ng/mL with a lower limit of quantification of 1 ng/mL in both human plasma and urine. The coefficient of variation of this assay precision was less than 13.7%, and the accuracy exceeded 92.0%. This method was successfully applied for pharmacokinetic study after oral administration of udenafil 100 mg to healthy Korean male volunteers.     

Simultaneous determination of creatinine and uric acid in human plasma and urine by micellar electrokinetic chromatography

High performance capillary electrophoresis using a buffer solution containing micelles of ionic surfactant (e.g. sodium dodecyl sulfate), called micellar electrokinetic chromatography, has been applied to the separation and simultaneous determination of creatinine and uric acid in human plasma and urine. The sample was introduced into the capillary by siphoning an appropriate volume of untreated plasma or urine spiked with an internal standard (antipyrine). Creatinine, uric acid, and antipyrine were separated mutually, and from other endogeneous components within 18 min. The calibration plots showed good linearity (correlation coefficient > 0.999) over the concentration range needed for clinical analysis. Standard addition tests indicated that the recoveries of creatinine and uric acid from urine samples ranged, respectively, from 97 % to 106 % and 97.4 % to 108 % with a coefficient of variation (C.V.) of 3.3 % (n = 5), and that those from plasma samples ranged, respectively, from 100 % to 112 % and 101 % to 107 % with a C.V. of 4.7 % (n = 5). The results were in agreement with those obtained by conventional methods.     

Highly sensitive method for the determination of JI-101, a multi-kinase inhibitor in human plasma and urine by LC-MS/MS-ESI: method validation and application to a clinical pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of JI-101 in human plasma and urine using LC-MS/MS-ESI in the positive-ion mode. The assay procedure involves extraction of JI-101 and alfuzosin (internal standard, IS) from human plasma/urine with a solid-phase extraction process. Chromatographic resolution was achieved on two Zorbax SB-C(18) columns connected in series with a PEEK coupler using an isocratic mobile phase comprising acetonitrile-0.1% formic acid in water (70:30, v/v). The total run time was 2.0 min. The MS/MS ion transitions monitored were 466.20 → 265.10 for JI-101 and 390.40 → 156.10 for IS. The method was subjected to rigorous validation procedures to cover the following: selectivity, sensitivity, matrix effect, recovery, precision, accuracy, stability and dilution effect. In both matrices the lower limit of quantitation was 10.0 ng/mL and the linearity range extended from ~10.0 to 1508 ng/mL in plasma or urine. The intra- and inter-day precisions were in the ranges 1.57-14.5 and 6.02-12.4% in plasma and 0.97-15.7 and 8.66-10.2% in urine. This method has been successfully applied for the characterization of JI-101 pharmacokinetics in cancer patients.     

A validated HPLC method with electrochemical detection for simultaneous assay of 5-aminosalicylic acid and its metabolite in human plasma

A high-performance liquid chromatographic method was developed, validated and applied to the simultaneous determination of 5-aminosalicylic acid (5-ASA) and its acetylated metabolite (acetyl-5-ASA) in human plasma. The method involves liquid-liquid extraction with methanol followed by isocratic reversed-phase chromatography on a Kromasil KR100 C(18) column with electrochemical detection. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from spiked human plasma samples. The effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers on retention of 5-ASA, acetyl 5-ASA and internal standard were investigated. Limits' of detection were 5 ng/mL for 5-ASA and 10 ng/mL for acetyl-5-ASA, respectively. The method can be used for supporting therapeutical drug monitoring and pharmacokinetic studies.     

基于抗原包被的微孔板式化学发光酶免疫分析法测定人尿中的雌三醇

将雌三醇-6-(O-羧甲基)肟(E3-6-CMO)与牛血清白蛋白(BSA)形成的偶联物E3-6-CMO-BSA物理吸附于聚苯乙烯微孔板孔内作为固相抗原,与雌三醇(E3)标准溶液或者水解尿样中待测E3通过竞争法进行免疫反应.以对碘苯酚增强的辣根过氧化物酶(HRP)催化鲁米诺-过氧化氢化学发光体系作为信号检测系统,建立了一种高通量、简便快速、灵敏稳定的化学发光酶免疫分析方法用于测定人尿中E3的含量.考察和优化了包被液的酸碱性、抗原包被浓度、酶标抗体稀释比例及用量、温育时间、化学发光底物用量及化学发光反应时间的影响.在最优实验条件下,方法的灵敏度为0.20ng/mL,批内和批间变异系数均在15%之内,低、中、高浓度加标水解尿样的平均回收率分别为107.9%、100.9%和91.2%.使用抗原包被法和抗体包被法同时对10份水解尿样进行测定,结果显示相关性良好,相关系数为0.9984,表明本方法可以满足临床检测的要求.     

Determination of imidol hydrochloride in human plasma and urine by high-performance liquid chromatography-tandem mass spectrometry and its application to clinical pharmacokinetic study

Imidol hydrochloride is a novel drug for the treatment of hepatitis B virus infection. A simple, special and sensitive solid‐phase extraction liquid chromatography–tandem mass spectrometry method for determination of imidol in human plasma and urine was developed for the first time and applied to a pharmacokinetic study. The chromatographic separation was achieved on a C₁₈ column (50 × 2.1 mm, 3.5 µm) using gradient elution with acetonitrile and water both containing 0.1% acetic acid at a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via a positive eletrospray ionization source. The mass transition pairs of m/z 517.8 → 325 and m/z 298 → 174 were used to detect imidol and the (?)‐clausenamide (internal standard), respectively. The retention times of imidol and (?)‐clausenamide were 2.5 and 2.7 min, respectively. Linearity, accuracy, precision, recovery, matrix effect, dilution test and stability were evaluated during method validation over the range of 0.2–500 ng/mL in human plasma and 0.5–500 ng/mL in urine. The method was successfully applied to a clinical pharmacokinetic study of imidol in healthy volunteers following oral administration. Copyright © 2011 John Wiley & Sons, Ltd.