Rapid determination of acetaminophen in physiological fluids by liquid chromatography using SDS mobile phase and ED detection

Acetaminophen is determined in serum and urine samples by a rapid, sensitive, and precise chromatographic method without any pretreatment step in a C18 column using a pure micellar mobile phase of 0.02M sodium dodecyl sulfate at pH 7. Acetaminophen is eluted in less than 5 min with no interference of the protein band. The use of electrochemical and UV detection is compared. Linearities (r > 0.999), as well as intra- and interday precision, are studied in the validation of the method. Limits of detection (LOD) are also calculated to be 0.56, 0.83, and 0.74 ng/mL in micellar solution, serum, and urine using electrochemical detection. The developed micellar liquid chromatographic method is useful for the quantitation of acetaminophen in serum and urine. Recoveries in the biological matrices are in the 98-107% range and results are compared with those obtained using a reference method. Drug excretion (in urine) and serum distribution are studied in several healthy volunteers, and no interference from metabolites is found. The developed procedure can be applied in routine analyses, toxicology, and therapeutic monitoring.     

Quantification of arecoline (areca nut alkaloid) in neonatal biological matrices by high-performance liquid chromatography/electrospray quadrupole mass spectrometry

A high-performance liquid chromatography (HPLC) method with mass spectrometric detection is described for determination of arecoline in newborn meconium, urine and cord serum, using pilocarpine as internal standard. The analytes were extracted from neonatal biological matrices with chloroform/isopropanol (95:5, v/v) at alkaline pH. Extracts were analyzed by HPLC coupled to an electrospray (ESI) interface and a quadrupole mass spectrometer. Chromatography was performed on a C(8) reversed-phase column using 10 mM ammonium acetate (pH 4.3)/acetonitrile (90:10, v/v) as mobile phase. The mass spectrometer was operated in selected ion monitoring mode. The method was validated over the concentration range 0.005-1.00 micro g/g meconium, 0.004-1.00 micro g/mL cord serum and 0.001-1.00 micro /mL urine. Mean recoveries ranged between 86.5 and 90.7% for arecoline in the different biological matrices, with precision always better than 10%. The quantification limits of arecoline were 0.005 micro g/g meconium, 0.004 micro g/mL cord serum, and 0.001 micro g/mL urine. The method was applied to the analysis of neonatal biological matrices to assess eventual fetal exposition to arecoline. Two newborns from Asian mothers who declared areca nut consumption presented arecoline in meconium with concentrations in the range 0.006-0.008 micro g/g; also the urine from one neonate tested positive for the drug.     

Determination of trazodone in urine and pharmaceuticals using micellar liquid chromatography with fluorescence detection

A simple and reliable liquid chromatographic procedure is described for the determination of trazodone in pharmaceutical formulations and urine samples. The optimized procedure uses fluorimetric detection, a C18 column and a micellar mobile phase of sodium dodecyl sulfate (SDS) and 1-butanol. The mobile phase selected for use was 0.2M SDS and 8% 1-butanol fixed at pH 3 with phosphate buffer. The total analysis time was 10 min. For the analysis of urine samples, one great advantage of the method is that no extraction step is required. The quantification limit was 9.5 ng mL(-1), ensuring the analysis of the drug in biological fluids. The procedure shows good accuracy, repeatability and selectivity. Repeatability and intermediate precision were tested for several concentrations of the drug. Good claim percentages were obtained in the analysis of pharmaceutical formulations. Calibration repeatability in urine matrix was also studied in the 0.06-22.4 microg mL(-1) range. Good recoveries were obtained from spiked urine samples. No interferences from common additives frequently administered with trazodone or from endogenous compounds in urine samples were found. The results show that the procedure is suitable for routine analysis of the drug.     

Analysis of 3‐methoxypterostilbene in biological fluids by high‐performance liquid chromatography: application to pre‐clinical pharmacokinetics

A method of analysis for 3‐methoxypterostilbene [trans‐3,3′5‐trimethoxy‐4′hydroxystilbene] in biological fluids is necessary to study pharmacokinetics. A novel and simple high‐performance liquid chromatographic method was developed for the determination of 3‐methoxypterostilbene in rat serum and urine. The internal standard, pinosylvin, was added to 0.1 mL serum or urine (serum proteins were precipitated with cold acetonitrile at ?20°C). Separation was achieved with a Phenomenex® C₁₈ (2) (5 µm, 250 × 4.60 mm) column with ultraviolet detection at 327 nm. The calibration curves in both matrices were linear ranging from 0.05 to 100 µg/mL, and the mean extraction efficiency was >99%. Precision of the assay for both matrices was <12% (RSD) and was within 13% for all points on the calibration curve. The limit of quantification for this method was 0.05 µg/mL. The assay was successfully applied to a preliminary study of 3‐methoxypterostilbene pharmacokinetics in a rat. Copyright © 2012 John Wiley & Sons, Ltd.     

超高效液相色谱-串联质谱法测定血浆与尿液中14种麻痹性贝类毒素北大核心CSCD

人体生物基质中麻痹性贝类毒素的检测对其引起的食物中毒诊断和救治具有重要意义。研究建立了超高效液相色谱-串联质谱法测定血浆、尿液中14种麻痹性贝类毒素的分析方法。实验比较了不同固相萃取柱的影响,优化了前处理条件和色谱条件,血浆样品采用0.2 mL水、0.4 mL甲醇、0.6 mL乙腈提取后直接上机测定,尿液样品采用0.2 mL水、0.4 mL甲醇、0.6 mL乙腈提取,聚酰胺(PA)固相萃取柱净化后上机测定。采用Poroshell 120 HILIC-Z色谱柱(100 mm×2.1 mm,2.7μm)对14种贝类毒素进行分离,流动相为含0.1%(v/v)甲酸的5 mmoL/L甲酸铵缓冲溶液和0.1%(v/v)甲酸乙腈溶液,流速为0.50 mL/min。在电喷雾模式(ESI)下进行正负离子扫描,采用多反应监测(MRM)模式检测,外标法定量。结果表明,对于血浆和尿液样品,14种贝类毒素分别在0.24~84.06 ng/mL范围内线性关系良好,相关系数均大于0.995。尿液检测的定量限为4.80~34.40 ng/mL,血浆检测的定量限为1.68~12.04 ng/mL。尿液和血浆样品在1、2和10倍定量限加标水平下平均回收率为70.4%~123.4%,日内精密度为2.3%~19.1%,日间精密度为4.0%~16.2%。应用建立的方法对腹腔注射14种贝类毒素小鼠血浆和尿液进行测定,20份血浆样本中检出含量分别为19.40~55.60μg/L和8.75~13.86μg/L。该方法操作简便,样品取样量少,方法灵敏度高,适用于血浆和尿液中麻痹性贝类毒素的快速检测。     

Mixed hemimicelles SPE based on CTAB-coated Fe3O4/SiO2 NPs for the determination of herbal bioactive constituents from biological samples

In this paper, a solid-phase extraction (SPE) method based on mixed hemimicelles of cetyltrimethyl ammonium bromide (CTAB) on silica-coated magnetic nanoparticles (MNPs) is developed for extraction and preconcentration of compounds from the biological samples. We selected rhein and emodin which are the major active anthraquinones of rhubarb as model analytes. A high performance liquid chromatography-fluorescence detection (HPLC/FLD) method was developed for the determination of rhein and emodin in urine and serum samples. The main factors influencing the extraction efficiency including the amount of surfactant, the concentration of MNPs, the shaking time and the desorption ability of organic solvents were investigated and optimized. No interferences were caused by proteins or endogenous compounds in urine and serum samples. Good linearities (r² > 0.9995) for all calibration curves were obtained, and the limits of detection (LODs) for rhein and emodin were 0.2 and 0.5 ng/mL in urine samples and 7 and 10 ng/mL in serum samples, respectively. Satisfactory recoveries (92.76-109.90% and 97.53-107.72% for rhein and emodin) in the biological matrices were achieved.     

Quantitative determination of the loop diuretic bumetanide in urine and pharmaceuticals by high-performance liquid chromatography with amperometric detection.

A high-performance liquid chromatographic method with amperometric detection has been developed for the determination of the diuretic bumetanide using a microBondapak C18 column. The mobile phase consists of a 50:50 acetonitrile-water mixture containing 5mM KH2PO4-K2HPO4 (pH 4.0). The compound is monitored at +1350 mV with an amperometric detector equipped with a glassy carbon working electrode. A liquid-liquid or solid-liquid extraction is done prior to chromatographic analysis in order to avoid the interferences found in the urine matrix. The percentages of recovery obtained are 71%+/-1% for liquid-liquid extraction and 84.2%+/-0.7% for solid-liquid extraction. The method developed has a linear concentration range from 50 to 499 ng/mL with a reproducibility in terms of relative standard deviation of 1.73% and 3.85% for a concentration level of 70 ng/mL and 237 ng/mL, respectively, and a detection limit of 0.25 ng/mL (3:1 signal-to-noise ratio). The method is applied to the determination of bumetanide in pharmaceutical formulations and urine obtained from hypertensive patients and healthy volunteers after the ingestion of a therapeutic dose of Fordiuran (1 mg bumetanide).     

Quantitative determination of the novel anticancer drug E7070 (indisulam) and its metabolite (1,4-benzenedisulphonamide) in human plasma, urine and faeces by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

E7070 (indisulam) is a novel anticancer drug currently undergoing clinical investigation. We present a sensitive and specific method for the quantitative determination of E7070 and its metabolite M1 (1,4-benzenedisulphonamide) in human plasma, urine and faeces. The analytes and their tetra-deuterated analogues, which were used as internal standards, were isolated from the biological matrix by solid-phase extraction with OASIS cartridges (0.5 mL plasma or 1 mL urine) and by liquid-liquid extraction with ethyl acetate at pH 5 (1 mL faecal homogenate). The analytes were separated on a C8 reversed-phase chromatographic column and analyzed using electrospray ionization and tandem mass spectrometric detection in the negative ion mode. The validated concentration ranges in plasma were 0.1-20 microg/mL for E7070 and 0.01-2 microg/mL for M1. In urine and faecal homogenate, a concentration range from 0.05-10 microg/mL or microg/g, respectively, was validated for both analytes. Validation of the plasma assay was performed according to the most recent FDA guidelines. The assay fulfilled all generally accepted requirements for linearity (r > 0.99, residuals between -8 and 10%), accuracy (-13.5 to 1.4%) and precision (all less than 11%) in the tested matrices. We investigated recovery, stability (working solutions at -20 degrees C and at room temperature, biological matrices at -20 degrees C, room temperature and after 3 freeze/thaw cycles; final extracts at room temperature) and robustness. All these parameters were found acceptable. This method is suited for mass balance studies or therapeutic drug monitoring, as demonstrated by a case example showing plasma concentrations and cumulative excretion of E7070 and M1 in urine and faeces. Furthermore, we show the presence of E7070 metabolites in patient urine.     

Separation and determination of 11 marker pteridines in human urine by liquid chromatography and fluorimetric detection

A simple liquid chromatographic method has been developed to achieve the complete separation and determination of a wide range of pteridinic compounds and creatinine (CREA) in urine samples, in just one run. The influences of mobile phase composition and buffer pH have been studied. The optimized mobile phase was composed of a Tris-HCl buffer (15 mmol/L) at pH 6.10 solution (eluent A) and a Tris-HCl buffer (15 mmol/L) at pH 6.40 solution (eluent B), in gradient mode. Analytes were determined by fluorimetric detection, exciting at 272 nm, and measuring the fluorescence emission at three wavelengths, 410, 445 and 465 nm. CREA, as a reference of metabolites excretion in urine, was determined by photometric detection at 230 nm. Pteridines detection limits varied from 0.2 to 6.1 ng/mL, and 0.2 g/mL for CREA. Calculated precision values expressed as RSD (%) varied from 1.1 to 5.9. Two different oxidation procedures for urine samples were optimized. The neopterin/biopterin ratios found were 0.98 and 0.86 for adults and children, respectively, by means of the alkaline iodide/iodine oxidation and 0.45 and 0.57 using neutral KMnO(4) oxidation.     

Improved high-performance liquid chromatographic method for the determination of 6-N,N,N-trimethyllysine in plasma and urine: biomedical application of chromatographic figures of merit and amine mobile phase modifiers

An internally standardized method for the determination of 6-N,N,N-trimethyllysine in human plasma, human urine, rat plasma, rat urine and hydrolyzed rat urine is described. This methylated amino acid and the procedural internal standard 6-N,N,N-trimethyllysine were isolated from the sample matrices using short ion-exchange columns and detected following high-performance liquid chromatography using a postcolumn reaction (o-phthalic-dicarboxaldehyde-2-mercaptoethanol) and fluorometric detection. The reliable detection limit for 6-N,N,N-trimethyllysine was 0.2 nmol/ml in 200 microliters of human plasma. The chromatographic separation exploits the unique properties of a novel tertiary amine mobile phase modifier, 3-(N,N-dimethylamino)-1,2-propanediol. The capacity factor and "Chromatographic Figures of Merit" (including peak asymmetry and relative system efficiency) were calculated for the chromatographic peak representing 6-N,N,N-trimethyllysine in over 2200 injections made while evaluating 900 biological specimens.     

Liquid Chromatographic Determination of 4-Amino-N-(2,6-dimethylphenyl)-benzamide in Rat Serum and Urine

Abstract

The compound 4-amino-N-(2,6-dimethylphenyl)-benzamide has shown potential as a new anticonvulsant. A method for the liquid chromatographic determination of serum and urine concentrations of the compound and its N-acetylated metabolite was developed for pharmacokinetic studies. Quantitation was achieved via UV detection at 275 nm following isocratic reversed phase (C₁₈) separation using a ternary solvent system of water:acetonitrile:acetic acid (60:39:1) at a flow rate of 1.5 mL/min. The compounds were isolated from a 50 μL sample of serum using solid phase extraction with prior protein precipitation. The compounds and internal standard were eluted from the extraction column with acetonitrile. Isolation from urine was achieved similarly with the exclusion of protein precipitation. The assay procedure is useful for the determination of concentrations of parent compound from 0.68 to 204.6 μg/mL.     

Quantitative determination of indapamide in pharmaceuticals and urine by high-performance liquid chromatography with amperometric detection.

A high-performance liquid chromatographic method with amperometric detection for the determination of the diuretic indapamide using a muBondapak C18 column is developed. The mobile phase consists of an acetonitrile-water mixture (45:55, 5 mM) in KH2PO4-K2HPO4 (pH 4.0). The compound is monitored at +1200 mV with an amperometric detector equipped with a glassy carbon working electrode. A liquid-liquid or solid-liquid extraction is performed prior to chromatographic analysis to avoid the interferences found in urine matrix. Percentages of recovery are 88.3 +/- 5.6 and 82.9 +/- 7.8 for liquid-liquid and solid-liquid extraction, respectively. The developed method has a linear concentration range from 25 to 315 ng/mL with a reproducibility in terms of relative standard deviation of 4% for a concentration level of 0.5 microgram/mL and a quantitation limit of 1 ng/mL. The method is applied to the determination of indapamide in tablets and urine obtained from hypertensive patients after the ingestion of Tertensif (indapamide 2.5 mg).     

Measurement of K-27, an oxime-type cholinesterase reactivator by high-performance liquid chromatography with electrochemical detection from different biological samples

K-27 is a bisquaternary asymmetric pyridinium aldoxime-type cholinesterase reactivator of use in the treatment of poisoning with organophosphorous esterase inhibitors. A sensitive, simple and reliable reverse-phase high-performance liquid chromatographic method with electrochemical detection was developed for the measurement of K-27 concentrations in rat brain, cerebrospinal fluid, serum and urine samples. Male Wistar rats were treated intramuscularly with K-27 and the samples were collected 60 min later. Separation was carried out on an octadecyl silica stationary phase and a disodium phosphate solution (pH 3.7) containing citric acid, octane sulphonic acid and acetonitrile served as mobile phase. Measurements were carried out at 30 degrees C at E(ox) 0.65 V. The calibration curve was linear through the range of 10-250 ng/mL. Accuracy, precision and the limit of detection calculated were satisfactory according to internationally accepted criteria. Limit of quantitation was 10 ng/mL. The method developed is reliable and sensitive enough for monitoring K-27 levels from different biological samples including as little as 10 microL of cerebrospinal fluid. The method--with slight modification in the composition of the mobile phase--can be used to measure a wide range of other related pyridinium aldoxime-type cholinesterase reactivators.     

Analysis of omeprazole and its main metabolites by liquid chromatography using hybrid micellar mobile phases

Omeprazole is a selective inhibitor of gastric acid secretion and is one of the most widely prescribed drugs internationally. A chromatographic procedure that uses micellar mobile phases of sodium dodecyl sulphate and propanol buffered at pH 7 and a C18 column is reported for the determination of omeprazole and its principal metabolites (omeprazole sulphone and hydroxyomeprazole) in urine and serum samples.In this work, direct injection and UV detection set at 305 nm was used. Omeprazole and its metabolites were eluted in less than 11 min with no interference by the protein band or endogenous compounds. Adequate resolution was obtained with a chemometric approach, in which the retention factor and shape of the chromatographic peaks were taken into account. The analytical parameters including linearity (r > 0.9998), intra- and inter-day precision (RSD, %: 0.6-7.9 and 0.14-4.7, respectively) and robustness were studied in the validation of the method for the three compounds. The limits of detection and quantification were less than 6 and 25 ng mL⁻¹, respectively. Recoveries in micellar medium, plasma and urine matrices were in the 98-102% range. Finally, the method was successfully applied to the determination of omeprazole and its metabolites in physiological samples. Omeprazole was also analysed in pharmaceutical formulations.     

Microanalytical high-performance liquid chromatographic assay for cefpirome in human milk and urine.

To permit the characterization of cefpirome disposition in lactating females, a previously published high-performance liquid chromatographic (HPLC) method for determining the drug in serum was adapted for use with milk and urine. This automated, microanalytical technique requires 50 microliters of biological matrix, which is subjected to an isopropanol extraction. Chromatography was accomplished using a microbore HPLC system, a reversed-phase C18 column and a mobile phase of 0.3% triethylamine in water (pH 5.1). Cefpirome and the internal standard (beta-hydroxypropyltheophylline) were monitored using UV detection at 240 nm and had retention times of 2.84 and 5.05 min, respectively. The method was linear up to 500 mg/l for both matrices and had a limit of detection of 0.6 mg/l. The interday variation (relative standard deviation) at concentrations of 5.0, 50.0 and 500.0 mg/l was consistently less than 5% in both urine and breast milk. The method was found to be free from interference by other commonly administered medications and readily adaptable for use in clinical investigations. The ease of sample preparation, small sample volume requirement, short chromatographic time, apparent lack of interferences, analytical sensitivity and high precision and accuracy make this method ideal for use in pharmacokinetic investigations involving the determination of cefpirome in human milk and urine.     

Method development and validation study for quantitative determination of nifedipine and related substances by ultra‐high‐performance liquid chromatography

A novel stability‐indicating reversed phase ultra‐high performance liquid chromatography (UPLC) coupled photodiode array gradient method was developed for determination of the nifedipine and related compounds. Furthermore, based on the chromatographic conditions and forced degradation studies performed through the development of the related substances method a UPLC isocratic method was validated for the determination of the assay of this active substance. An Acquity Shield RP₁₈ (50 × 3.0 mm 1.7 µm) column was used for separation of nifedipine and its five potential impurities within 11 min, which is 5‐fold less than the official method. A mobile phase consisting of 10 mm ammonium formate (pH 4.5) and methanol, delivered at a flow rate 0.5 mL/min, was employed to achieve a minimum resolution of 2.0 for all consecutive pairs of compounds. The precision value expressed as percentage relative standard deviation for method repeatability and reproducibility was <5.0%. The recoveries for all the related compounds were in the range of 99–105.0%. Linearity was found to be acceptable over the concentration range of 0.25–1.5 µg/mL for nifedipine and its impurities. The limit of quantification for nifedipine was 0.05 µg/mL, which is much less than the European Pharmacopoeia method. Copyright © 2014 John Wiley & Sons, Ltd.     

Direct determination of mitoxantrone and its mono- and dicarboxylic metabolites in plasma and urine by high-performance liquid chromatography

The simultaneous isolation and determination of mitoxantrone (Novantrone) and its two known metabolites (the mono- and dicarboxylic metabolites) were carried out using a high-performance liquid chromatographic (HPLC) system equipped with an automatic pre-column-switching system that permits drug analysis by direct injection of biological samples. Plasma or urine samples were injected directly on to an enrichment pre-column flushed with methanol-water (5:95, v/v) as the mobile phase. The maximum amount of endogenous water-soluble components was removed from biological samples within 9 min. Drugs specifically adsorbed on the pre-column were back-flushed on to an analytical column (Nucleosil C18, 250 X 4.6 mm I.D.) with 1.6 M ammonium formate buffer (pH 4.0) (2.5% formic acid) containing 20% acetonitrile. Detection was effected at 655 nm. Chromatographic analysis was performed within 12 min. The detection limit of the method was about 4 ng/ml for urine and 10 ng/ml for plasma samples. The precision ranged from 3 to 11% depending on the amount of compound studied. This technique was applied to the monitoring of mitoxantrone in plasma and to the quantification of the unchanged compound and its two metabolites in urine from patients receiving 14 mg/m2 of mitoxantrone by intravenous infusion for 10 min.     

High-performance liquid chromatographic determination of BRB-I-28, a novel antiarrhythmic agent, in dog plasma and urine.

A sensitive reversed-phase high-performance liquid chromatographic (HPLC) technique with ultraviolet detection has been developed to determine the concentration of BRB-I-28 (I), a novel antiarrhythmic agent, in dog plasma and urine. The mobile phase was acetonitrile-methanol-37.5 mM phosphate buffer, pH 6.8-triethylamine (50:50:75:0.1, v/v). The compound was extracted from dog plasma and urine with chloroform after alkalinization with sodium hydroxide. The extraction recovery was 83% from plasma and 84% from urine. Good linearity (r > 0.996) was observed throughout the ranges 0.1-12.0 micrograms/ml (plasma) and 0.1-8.0 micrograms/ml (urine). Intra- and inter-assay variabilities were less than 4%. The lower limit of quantitation was 0.08 microgram/ml in either plasma or urine. HPLC analysis of plasma and urine samples from a dog treated with I has demonstrated that the method was accurate and reproducible.     

Determination of tazobactam and piperacillin in human plasma, serum, bile and urine by gradient elution reversed-phase high-performance liquid chromatography

A gradient elution high-performance liquid chromatographic method is described for the analysis of the beta-lactamase inhibitor tazobactam (YTR-830H) and a semi-synthetic parenteral penicillin, piperacillin, in human plasma, serum, bile and urine. The assay for plasma, serum and bile involves deproteinization with acetonitrile and the removal of lipids with dichloromethane; urine is diluted with buffer. Separation and quantitation are achieved using a mobile phase based on ion-suppression chromatography on a C18 reversed-phase column with ultraviolet detection at 220 nm. The limit of quantitation for both compounds is 1.0 microgram/ml in plasma, serum and bile using a 0.2-ml sample and 50.0 micrograms/ml in urine using a 0.1-ml sample. The method has been validated by preparing and analyzing a series of fortified samples (range 1.0-200 micrograms/ml for each compound in plasma, serum and bile and 50.0-10,000 micrograms/ml for each compound in urine). Excellent linearity, accuracy, precision and recovery were obtained. The method was not interfered with by other endogenous components, nor by other commonly administered antibiotics such as amoxicillin, mezlocillin, cefometazole and cefotaxime. The assay has been successfully applied to the analysis of samples from pharmacokinetic studies in man and animals.