Quantitation of Serotonin in Human Plasma,Serum and Cerebrospinal Fluid Samples by HPLC-EC Using 6-Hydroxytryptamine as an Internal Standard

Abstract

A simple method for sample clean up and concentration of serotonin (5-HT) in biological samples, such as human cerebro-spinal fluid and serum, is described. To the sample 6-hydroxy-tryptamine (6-HT) is added as an internal standard and it is then absorbed either on C₁₈ SEP-PAK cartridge or Biorex-70 short column. 5-HT and 6-HT are then eluted from the column with methanolic formic acid. After evaporation, the residue is dissolved in the mobile phase and an aliquot is used for LC-EC quantitation.     

HPLC Analysis of Cefmetazole and Nocardicins A and E in Human Serum and Urine Using Solid-Phase Extraction

Abstract

A method for extraction and quantification of cefmetazole and nocardicins A and E in serum and urine samples is described in this paper. Sample pretreatment is carried out using solid-phase extraction cartridges, resulting in very high extraction recoveries of these β-lactam antibiotics. The procedure, which prepares biological fluids for reversed-phase high-performance liquid chromatographic analysis is convenient, rapid and reproducible. An water-methanol-acetic acid mobile phase was used with benzotriazole as an internal standard. The detection limit was 0.2 μg/ml at 280 nm.     

Gentamicin Determination in Biological Fluids by HPLC,Using Tobramycin as Internal Standard

Abstract

An improved procedure to quantify gentamicin in biological fluids is presented. The antibiotic is isolated from plasma and urine by using a silica column, and measured by reversed-phase chromatography. Pre-column derivatization with o-phthalaldehyde to form fluorescent products for detection is used. The method can accurately measure 0.3 mg of gentamicin per liter, and standard curves showed a linear response in plasma and urine at concentrations ranging from 0 to 10 mg/liter. The different processes described in the literature for aminoglycoside isolation are discussed and evaluated with reference to the present results. Moreover, the     

Micro Assay for Malondialdehyde in Human Serum by Extraction-Spectrophotometry Using an Internal Standard

Malondialdehyde (MDA) is a widely accepted biomarker of lipid peroxidation. Thus, the measurement of MDA in clinical samples is useful in the evaluation of oxidative stress. In this study, a micro-extraction-spectrophotometric assay was developed, based on the formation of MDA–thiobarbituric acid (TBA) adduct. To enhance the analytical performance, Erioglaucine A was used as an internal standard (IS), and the first derivative spectra were obtained. The volume of serum sample was 20µL and the total volume of aqueous phase 420µL (200µL of 0.6% TBA in acetic acid, pH 2.5 and 200µL of 6.25·10^–4% IS). The extraction of adduct and IS was carried out with 300µL of aliquat 336 (0.06%) in ethyl acetate. The analytical signal S was defined as the ratio between the first derivative absorbances measured at 543.1nm (adduct) and 644.4nm (IS). In the calibration range up to 10µmolL^–1 MDA, the linear regression coefficient was 0.9998. The quantification limit was 0.19µmolL^–1 and the CV values for 2µmolL^–1 and 5µmolL^–1 MDA, respectively, were 0.8% and 0.7%. The procedure was applied to the analysis of diabetic sera and the results compared with those obtained by HPLC (derivatization with 2,4-dinitrophenylhydrazine). Lower HPLC results (about 15%) indicated that interferences from other TBA reactive substances had not been completely eliminated by the extraction procedure and derivatization of spectral data. On the other hand, the micro-procedure presents important advantages: it is simple, precise and environmentally friendly (small amounts of reagents), which makes it readily adaptable to the analysis of large sample series. The feasibility of micro-assay in the evaluation of lipid peroxidation status was demonstrated in the analysis of 156 serum samples from diabetic patients divided into three groups according to the stage of development of typical complications.     

An HPLC Method for the Determination of Theophylline and Its Metabolites in Serum and Urine

Abstract

A urine and a serum assay have been developed to quantitate theophylline and its major metabolites:1,3-dimethyluric acid, 3-methylxanthine and 1-methyluric acid. Reverse phase chromatography follows a serum acetone extraction procedure and a urine anion exchange clean-up procedure. Lower limits of sensitivity are 0.04 μg/ml for serum metabolites and 1 μg/ml for urine metabolites. Both assays are free of interference from endogenous substances. These assays have been tested successfully in pharmacokinetic and metabolic studies of theophylline.     

Validation of an HPLC Method for Determination of Cefepime (a Fourth-Generation Cephalosporin). Determination in Human Serum, Cerebrospinal Fluid, and Urine. Pharmacokinetic Profiles

A high-performance liquid-chromatographic method with detection at 256 nm has been developed and validated for analysis of cefepime in several biological matrices. Serum samples were deproteinized with acetonitrile and extracted once with dichloromethane. For urine and cerebrospinal fluid samples, only a microfiltration step was necessary. The method was validated in accordance with the recommendations of the International Conference on Harmonization (ICH), the Food and Drug Administration (FDA), and the Center for Drug Evaluation and Research (CDER). The method was used to determine levels of the drug in the serum, cerebrospinal fluid, and urine of twelve patients treated with Maxipime. The results obtained were compared with those from previously published HPLC methods.     

高效液相色谱化学发光检测人体血清及尿样中的盐酸肾上腺素

研究发现,盐酸肾上腺素在碱性条件下能显著增强铁氰化钾-鲁米诺化学发光强度。基于此建立了新的测定肾上腺素的方法。本方法以C18反相键合相为色谱柱,用0.01 mol/L邻苯二甲酸氢钾-甲醇(92∶8,V/V)为流动相,实现了对人体血清及尿样中盐酸肾上腺素的分离与测定。在最适宜条件下,方法的线性范围为10~5000μg/L;检出限为4.0×10-6g/L;相对标准偏差为3.0%(n=11)。     

Determination of Celecoxib, Rofecoxib, Sodium Diclofenac and Niflumic Acid in Human Serum Samples by HPLC with DAD Detection

A reverse-phase high-performance liquid chromatographic method has been developed for the separation and simultaneous determination of two COX-2 inhibitors, celecoxib and rofecoxib, in addition to two well-known non-steroidal anti-inflammatory drugs (NSAIDs), sodium diclofenac and niflumic acid in human serum samples. Good chromatographic separation was achieved using a C18 bonded silica column applying a gradient with acetronitrile and water, from 15 to 60% acetonitrile. The mobile phase contained 0.1% trifluoroacetic acid as an organic modifier. Detection was made using a diode array detector (DAD) and the analytical parameters were established at the wavelength maximum in the UV spectrum of each drug. Linearity was studied up to 100.0 mg L⁻¹. Calibration functions, quantification and detection limits, intra- and inter-day reproducibility and accuracy were estimated for each drug. Solid phase extraction was needed to separate and concentrate the drugs from human serum samples. The method was successfully applied to determine the drugs in human serum samples at levels of 1.0 mg L⁻¹.     

Determination of Piribedil in Human Serum, Urine and Pharmaceutical Dosage Form by LC-DAD

A rapid, selective and sensitive reversed-phase liquid chromatographic (LC) method was developed for the determination of piribedil in human serum, urine and pharmaceutical dosage form. LC analysis was carried out using reversed-phase isocratic elution with a C₁₈ column and a mobile phase of 0.01 M phosphate buffer-acetonitrile (50:50, v/v). The chromatograms showed good resolution and sensitivity with no interference of human serum and urine. Piribedil concentrations were determined using diode array detection at 240 nm. Sildenafil citrate was used as internal standard. The limit of quantification (LOQ) and limit of detection (LOD) concentrations were 107.2 and 321.6 pg mL^?1, 96.6 and 290.4 pg mL^?1, 161.7 and 53.9 pg mL^?1 for urine, serum and pharmaceutical dosage forms, respectively. The method was validated for its linearity, precision and accuracy and applied to the tablets, urine and human serum. In addition, the results were compared to those obtained from UV-spectrophotometry.     

分散液相微萃取-高效液相色谱法同时测定尿样中2,5-己二酮、苯酚及邻甲酚

提出了分散液相微萃取-高效液相色谱法同时测定尿样中2,5-己二酮、苯酚及邻甲酚的含量。优化的试验条件如下:1衍生试剂为2,4-二硝基苯肼;2反应时间为20min;3反应温度为70℃;4萃取剂三氯甲烷的用量为0.3mL;5萃取时间为10min。以C18色谱柱为固定相,用不同比例的甲醇-水溶液为流动相进行梯度洗脱,用紫外检测器测定。2,5-己二酮、苯酚及邻甲酚在一定的质量浓度范围内与其峰面积呈线性关系,方法的检出限(3S/N)在0.3~0.7μg·L-1之间。加标回收率在89.1%~104%之间,测定值的相对标准偏差(n=7)在3.0%~4.6%之间。     

Determination of Tryptophan in Lysozyme and Lysozyme Hydrolysate by Chiral LC Using d-Tryptophan as Internal Standard

In the present study, a new LC method is described for the quantitation of tryptophan (Trp) in lysozyme and enzymatic lysozyme hydrolysate. To compensate for partial breakdown of Trp during hydrolysis with 4 M methanesulfonic acid, an enantiomer dilution method was developed. The method makes use of free d-Trp or a d-Trp-containing dipeptide as internal standard for the quantitation of l-tryptophan in these matrices. After acid hydrolysis in 4 M methanesulfonic acid, LC analysis is performed on a Crownpak CR chiral column in combination with fluorescence detection. Optimum time and temperature for the acid hydrolysis were investigated in order to obtain complete hydrolysis of the source materials. A comparison of the l-Trp recoveries was made for d-Trp and Gly-d-Trp as internal standards. By choosing a hydrolysis time of 150 min at 150 °C, 93% recovery of l-Trp from lysozyme was achieved. Under these conditions, no racemization occurred. When choosing d-Trp as internal standard, a direct LC method for l-Trp in lysozyme and enzymatic lysozyme hydrolysate was established without the need for pre-column derivatization and without the need to use Trp protecting agents during acid hydrolysis.     

Determination of Tryptophan in Lysozyme and Lysozyme Hydrolysate by Chiral LC Using d-Tryptophan as Internal Standard

In the present study, a new LC method is described for the quantitation of tryptophan (Trp) in lysozyme and enzymatic lysozyme hydrolysate. To compensate for partial breakdown of Trp during hydrolysis with 4 M methanesulfonic acid, an enantiomer dilution method was developed. The method makes use of free d-Trp or a d-Trp-containing dipeptide as internal standard for the quantitation of l-tryptophan in these matrices. After acid hydrolysis in 4 M methanesulfonic acid, LC analysis is performed on a Crownpak CR chiral column in combination with fluorescence detection. Optimum time and temperature for the acid hydrolysis were investigated in order to obtain complete hydrolysis of the source materials. A comparison of the l-Trp recoveries was made for d-Trp and Gly-d-Trp as internal standards. By choosing a hydrolysis time of 150 min at 150 °C, 93% recovery of l-Trp from lysozyme was achieved. Under these conditions, no racemization occurred. When choosing d-Trp as internal standard, a direct LC method for l-Trp in lysozyme and enzymatic lysozyme hydrolysate was established without the need for pre-column derivatization and without the need to use Trp protecting agents during acid hydrolysis.

     

Determination of Adrenaline by Atomic Absorption Spectrometry Using Copper as Probe Reagent in Pharmaceutical and Serum Samples

In this paper, an atomic absorption spectrometric method has been established to determine adrenaline using Cu(II) as probe reagent. In the presence of SCN^?, it has been demonstrated that Cu(II) is reduced to Cu(I) by adrenaline at pH 6.0, and the in situ formed Cu(I) reacts with SCN‐ to form the white emulsion of CuSCN. According to the amount of residual Cu(II) which is measured by atomic absorption spectrometry, the amount of adrenaline can be determined indirectly. Beer's law is obeyed in the range of adrenaline concentration of 0.080‐6.00 mg mL‐1, and the linear regression equation is A = 0.20342 ‐ 0.02756C (μg mL^?1) with a correlation coefficient of 0.9998. The detection limit is 0.033 μg mL^?1 and R.S.D. is 0.56%. This method has been successfully applied to determine adrenaline in pharmaceutical and serum samples with satisfactory results.     

Direct Analysis of Phenol, Catechol and Hydroquinone in Human Urine by Coupled-Column HPLC with Fluorimetric Detection

Phenol, catechol, and hydroquinone, are urinary end-products of the metabolism of benzene, nutrients, drugs, and endogenous substances. Recent research demonstrated that phenol, catechol, and hydroquinone, may have themselves a role in the carcinogenicity of benzene and in mechanisms that lead to leukemia. In this respect there is the need of rapid, low-cost, and possibly direct methods to quantitate these phenolic metabolites. Three single-residue coupled-column HPLC methods with fluorimetric detection (LC-LC-FLD) are described for the direct quantitation of phenol, catechol, and hydroquinone, in human urine. After enzymatic hydrolysis of the corresponding beta-glucuronoconjugates and sulfates, urine was directly injected into the LC-LC analyzer. The LC-LC-FLD procedure allowed base-to-base separation of the target compounds from urine interferents and good linearity (r² = 0.998) within the ranges studied (0.5–50 mg L⁻¹ for phenol, 0.35–35 mg L⁻¹ for catechol and 0.2–10 mg L⁻¹ for hydroquinone). Despite the high background levels of these metabolites in human urine, within- and inter-session precision expressed as RSD% was better than 20% on spiked and on authentic urine samples obtained from benzene-exposed workers. Accuracy expressed as the recovery ratio between measured and nominal concentration in spiked urine was comprised between 93% and 115% for the three metabolites. The column switching system was fully automated and computer-controlled, and was applied to the determination of phenol, catechol, and hydroquinone in urine samples showing a sample throughput of at least 20–30 samples per day.Revised: 21 February and 7 April 2005     

Stereoselective Determination of Ornidazole Enantiomers in Human Plasma and Urine Samples by Chiral LC: Application to Pharmacokinetic Study

A stereoselective liquid chromatographic method to determine the enantiomers of ornidazole in human plasma and urine has been developed and validated. After addition of the internal standard (naproxen), samples were acidified and extracted with diethyl ether. The separation was performed on a Chiralcel OB-H column, using hexane-ethanol- glacial acetic acid (94:6:0.08, v/v) as the mobile phase. The method was validated for specificity, linearity, sensitivity, precision, accuracy and stability. For each enantiomer of ornidazole, linear calibration curves were obtained over the concentration range of 0.16–20 μg mL^?1 in plasma and 0.32–20 μg mL^?1 in urine. For both enantiomers of ornidazole in plasma and urine, the coefficient of variation for precision were consistently less than 12% and accuracy were within ±14% in terms of relative error. Application of the method to a preliminary pharmacokinetic study showed that this validated method was qualified for the direct determination of ornidazole enantiomers in human plasma and urine.     

Stereoselective Determination of Ornidazole Enantiomers in Human Plasma and Urine Samples by Chiral LC: Application to Pharmacokinetic Study

A stereoselective liquid chromatographic method to determine the enantiomers of ornidazole in human plasma and urine has been developed and validated. After addition of the internal standard (naproxen), samples were acidified and extracted with diethyl ether. The separation was performed on a Chiralcel OB-H column, using hexane-ethanol- glacial acetic acid (94:6:0.08, v/v) as the mobile phase. The method was validated for specificity, linearity, sensitivity, precision, accuracy and stability. For each enantiomer of ornidazole, linear calibration curves were obtained over the concentration range of 0.16–20 μg mL⁻¹ in plasma and 0.32–20 μg mL⁻¹ in urine. For both enantiomers of ornidazole in plasma and urine, the coefficient of variation for precision were consistently less than 12% and accuracy were within ±14% in terms of relative error. Application of the method to a preliminary pharmacokinetic study showed that this validated method was qualified for the direct determination of ornidazole enantiomers in human plasma and urine.

     

Simultaneous Determination of Abacavir,Efavirenz and Valganciclovir in Human Serum Samples by Isocratic HPLC-DAD Detection

A rapid, sensitive, and specific reverse phase high performance liquid chromatography with diode array detection procedure for the simultaneous determination of abacavir, efavirenz and valganciclovir in spiked human serum is described. Separation was performed on a 5 μm Waters Spherisorb column (250 × 4.6 mm ID) with acetonitrile: methanol:KH₂PO₄ (at pH 5.00) (40:20:40 v/v/v) isocratic elution at a flow rate of 1.0 mL min⁻¹. Calibration curves were constructed in the range of 50–30,000 ng mL⁻¹ for abacavir and efavirenz, and 10–30,000 ngmL⁻¹ for valganciclovir in serum samples. The limit of detection and limit of quantification concentrations of the HPLC method were 3.80 and 12.68 ng mL⁻¹ for abacavir, 2.61 and 8.69 ng mL⁻¹ for efavirenz, 1.30 and 4.32 ng mL⁻¹ for valganciclovir. The method has been applied, without any interference from excipients or endogenous substances, for the simultaneous determination of these three compounds in human serum.

     

十二烷基苯磺酸钠增敏乙基紫共振光散射法测定人尿中痕量1-羟基芘

基于乙基紫(EV)与1-羟基芘(1-OHP)反应形成离子缔合物,导致体系的共振光散射(RLS)增强,建立了测定1-OHP的共振光散射法.在pH 8.0Tris-HCl缓冲介质中,最大RLS峰位于396 nm,其强度与1-OHP浓度成线性关系.加入表面活性剂十二烷基苯磺酸钠(SDBS),体系的灵敏度可提高两倍以上.方法的线性范围为4.0～982 μg·L-1,相关系数r=0.999 4,检出限1.2 μg·L-1,相对标准偏差为5.8%～7.9%,平均回收率为94%(n=6).已成功用于人尿中痕量1-OHP的测定,结果满意.     

A Validated HPLC Method with Fluorescence Detection for the Determination of Droperidol in Pharmaceutical Tablets,Human Serum,and Human Milk

A sensitive and simple HPLC method with fluorimetric detection has been developed for determination of droperidol in pharmaceutical tablets, human serum, and human milk. Chromatography was performed on a 100 mm × 3 mm i.d. C₁₈ column with methanol–water, 30:70 (v/v), pH 3.5, as mobile phase at a flow-rate of 0.8 mL min⁻¹. The injection volume was 5 μL and detection was by monitoring emission at 324 nm after excitation at 283 nm. Droperidol and p-hydroxybenzoic acid (internal standard) eluted after 5.3 and 6.1 min, respectively. The method was validated over the concentration range 1.14 × 10⁻⁷ to 9.12 × 10⁻⁶ M. Selectivity was good and the limits of detection and quantitation of the method were approximately 3.54 × 10⁻⁸ and 1.07 × 10⁻⁷ M, respectively, corresponding to 13 and 40 ng mL⁻¹. The applicability of the method to determination of droperidol in pharmaceuticals, human serum, and human milk was demonstrated.