A specific sensitive HPLC method for determination of plasma dopamine

Summary We describe here a sensitive, selective and rapid method to quantitate plasma catecholamines, especially dopamine, using high-performance liquid chromatography with electrochemical detection. This method requires a 10-minute run time and has a threshold for detection of 2 picograms, (10pg/ml).A number of commonly employed mobile phases for catecholamine analysis have been tested and have failed to detect dopamine in biological samples. Neither acetonitrile (3–7%) or methanol, (5–8%) in the mobile phase has produced consistently interpretable data either due to inability to detect or interference from co-eluting substances. Optimal detection was achieved with a mobile phase containing sodium acetate (6.8g), citric acid (5.9g), EDTA (48mg), di-n-butylamine (270l), Na-1-octane sulfate (850mg), methanol (100 ml) (amounts refer to 1 liter aqueous solution) (pH 4.3). The mobile phase was passed through a Waters 5 resolve C₁₈ column using a Waters 590 pump and m460 electrochemical detector and 740 data module, Flow rate was 0.9ml/min. Using this method, normal values in human and swine left ventricular myocardium and human and swine plasma have been established for norepinephrine, epinephrine, and dopamine.     

Highly sensitive method for the determination of a new anthracycline: Pirarubicin

Summary A new and highly sensitive HPLC method for the simultaneous determination of pirarubicine (THP-doxorubicin) and its metabolites, adriamycin and adriamycinol, in human plasma, is described. Samples were treated by liquid-liquid extraction, the organic phase removed and the residue dissolved in methanol. Separation was on a Lichrocart Supersher RP 8 column, (250×4 mm) 4 m, with a mobile phase of acetonitrile/methanol/formate-buffer.     

A highly sensitive method for the determination of adriamycin and adriamycinol in human plasma using HPLC with fluorimetric detection

Summary A simple and very sensitive HPLC method, for the simultaneous determination in human plasma of adriamycin and its metabolite adriamycinol, is described. Plasmas from patients were stored frozen. Thawed samples were extracted by absorption of anthracyclin onto a small C18 column. After evaporation of the eluate and reconstitution of the residue with methanol (100L), 30 to 40L of the mixture were injected into the chromatograph. Separation was obtained using an RP 8 column with a mobile phase of formate buffermethanol-acetonitrile (502327, v/v). A spectrofluorimeter was used as detector. The limit of sensitivity of the assay was 50 pcg/ml of plasma.     

HPLC method for determination of cefuroxime in plasma

Summary This paper describes an HPLC method for the determination of cefuroxime in human plasma. The method uses solid phase extraction (SPE) and has acceptable sensitivity, precision and accuracy. The limit of quantification in plasma samples is 0.1 μg mL⁻¹. Calibration curves were linear within 0.1–20 μg mL⁻¹, with mean correlation coefficient of 0.9982. Mean inter day precision and accuracy were 7.8% and 6.4%, respectively. The method was applied to determine cefuroxime levels in patients receiving cefuroxime, 3 time per day.     

Modified polystyrene,stationary phases for the separation of different aromatic hydrocarbon types by HPLC

Summary One possible method of characterizing chemicall-modified stationary phases is to describe their chromatographic properties. In this study we investigated several chemically-modifiedpolystyrene-divinylbenzene packings for the separation of nitroarenes, polycyclic aromatic hydrocarbons and aminosubstituted aromatic hydrocarbons. Chromatography was carried out on commercially available polymer stationary phases, for example C-18 or vinylpyridine modified polystyrene. In addition, a chemically-immobilized polymer packing was prepared by introducing nitrogroups, which were established by elemental analysis and infrared spectroscopy.     

Development and validation of a stability-indicating HPLC method for the determination of degradation products in dipyridamole injection

Summary The development and subsequent validation of an isocratic high-performance liquid chromatographic (HPLC) procedure employing ultraviolet (UV) detection for the determination of degradation products in Dipyridamole Injection is reported. The development of this assay involved the evaluation of several factors including buffer type, ionic strength, pH, organic composition, and column type. The described method is simple, reproducible, accurate, and selective. The precision, relative standard deviation (RSD), amongst five sample preparations for total degradation products was not more than (NMT) 10.2 %, while the individual degradation products were NMT 12.1%. Intermediate precision, as determined from fifteen sample preparations, generated by two Analysts on different HPLC systems over three days, exhibited an RSD for total and individual degradation products of 8.2 % and NMT 27.5 %, respectively. The mean absolute recovery of dipyridamole using the described method is 102.1±1. 9%, (mean±SD, n=12) over the concentration range of 0.03 % to 5.0 % of its label claim of 5 mg mL⁻¹. The limit of detection and limit of quantitation were 0.1 and 0.3 μg mL⁻¹, respectively. The linearity of the peak response was verified with respect to dipyridamole concentration over a range of 0.3 and 50 μg mL⁻¹ (0.03 % to 5.0 % label claim). The Standard and Assay Preparations are stable for up to 48 hours at room temperature. The selectivity was evaluated by subjecting the finished product (Dipyridamole Injection) to thermal, acidic, basic, oxidative and fluorescent radiation stress conditions. No interference in the analysis of degradation products was observed, showing the method is stability-indicating.     

Simple and rapid HPLC method for simultaneous determination of multiple antiepileptic drugs in human serum

Summary A very rapid, sensitive and reproducible HPLC method was developed for simultaneous determination of eight anti-epileptic drugs (AEDs): lamotrigine, primidone, ethosuximide, sulthiame, felbamate, phenobarbital, carbamazepine, phenytoin and oxcarbazepine-metabolite (10-hydroxy-carbazepine) in human serum. Sample purification requires only protein precipitation with an appropriate reagent. Separation was by reversed-phase HPLC, using a C18 column, 20% acetonitrile and 40 mM phosphoric acid buffer as mobile phase. Column temperature was set at 50°C, and measurement was by UV detection at 205 nm. The inter and intra-assay coefficients of variation (CV) ranged 1.13–7.10% and 1.14–8.49%, respectively. The absolute (measured) and relative (analytic) recoveries of the drugs ranged 96.7%–104.4% and 97.3%–106.1%, respectively. No interference with other common antiepileptic drugs and analgesics were observed. The method requires only 100 μl serum or less. It is very fast (sample preparation and analysis time approx. 23 min for all 9 AEDs), and suitable for routine clinical use, especially for epileptic patients on polytherapy.     

An improved high-performance liquid chromatography method for the determination of homocysteine in human plasma

Summary Elevated plasma homocysteine is, a known risk factor in arteriosclerotic vascular disease. To measure homocysteine in a large number of samples, we have developed a rapid, simple, robust and inexpensive reversed-phase HPLC method for routine analysis. Mercaptopro-pionylglycine was used as the internal standard and an external calibration in plasma was performed. Improvement was achieved by the use of gradient elution (using a sodium acetate buffer and methanol) resulting in a higher number of samples analyzed per day. Plasma samples were reduced with tributylphosphine and the proteins were precipitated with perchloric acid before addition of internal standard. The analytes were derivatized by use of 7-fluorobenzofurazone-4-sulfonic acid ammonium salt. For calibration human plasma was spiked with nine different concentrations of homocysteine (range 2–50 μmol L⁻¹). The inter-assay precision of replicate (n=29) analysis of the concentration of homocysteine in a sample of pooled plasma was 3.0%. The limit of detection, defined as three times the signal-to-noise ratio, was 0.25 μmol L⁻¹. The linearity of the assay was confirmed for a plasma concentration range of 2–2000 μmol L⁻¹. The variation of duplicate analyses of 842 plasma samples was 2.6±1.7%.     

HPLC法测定人尿中尿酸含量

建立了一种人体尿液中尿酸含量的高效液相色谱测定方法.采用ACE5 AQ亲水色谱柱,pH3.2的乙酸水溶液为流动相,检测波长280nm.尿酸含量在7.1～224.6μg/mL范围内线性关系良好,平均加样回收率为99.7%～100.5%,RSD小于1.4%.将该法用于健康人和肝硬化病人尿液样本的测定,两类样本中尿酸含量无显...     

Evaluation of a spectrofluorimetric method for the selective determination of thalidomide in pharmaceutical tablets, urine and blood serum

A spectrofluorimetric analytical methodology for the determination of thalidomide in pharmaceutical formulations, blood serum and urine was developed and evaluated in terms of sensibility and selectivity. Efforts were spent on the maximization of thalidomide fluorescence signal and on the increasing of selectivity when analyzing complex samples, specially, in samples containing sulfanilamide, a strongly fluorescent concomitant that can be used in association with thalidomide. Maximum signal was observed with optimized composition of the solvent system and pH. Approaches to enable selective determination of thalidomide in the presence of sulfanilamide were tested and compared. Better results were achieved using the combined use of acidic medium and UV treatment. For urine and blood serum samples, a solid phase extraction (on a C₁₈ cartridge) was found to be practical, enabling good recovery results. Limit of detection (3S_b/m) was estimated to be 1.2 μg l⁻¹, approximately 100 times better than the ones reported for the routine UV absorption HPLC methods. This low-cost spectrofluorimetric methodology was found to be very simple and enabled excellent recoveries when analyzing analyte spiked biological fluids as well as thalidomide based commercial and laboratory made pharmaceutical formulations.     

Capillary electrophoresis as an alternative to HPLC for determination of honey flavonoids

Summary The general objective is to provide an alternative methodology based on capillary electrophoresis (CE) to characterize flavonoids from honey and hence determine its botanical origin. The specific objective is to compare the separation of flavonoids by CE with those achieved by HPLC to assess CE as an alternative technique for the determination of honey flavonoids. Fourteen different flavonoids isolated from honey were analysed by MECC and compared to the HPLC separations. It was difficult to find specific experimental conditions to separate all the flavonoids from honey in a single MEKC run. Three chromatographic conditions are optimized and, depending on the flavonoid markers sought in honey, the appropriate detection method should be chosen. Compared to the HPLC results, it is clear that CE could be an alternative technique in honey flavonoids analysis and particularly in the study of its geographical and floral origin.     

An optimized direct method for the determination of carboxylic acids in beverages by HPLC

Summary A direct method for the simultaneous determination of tartaric, malic, lactic, acetic, citric, shikimic, fumaric and succinic acids in fruit juices and wines by isocratic reversed phase HPLC is reported.The variables (pH, ionic strength, flow and temperature) have been optimized by a modification of the original simplex method. The separation factor (s) and calibrated resolution product (r^*) have been used as criteria for selectivity optimization. After validation, the method has been applied to the determination of carboxylic acids in apple, orange and lemon juices, white and red wines and musts during the fermenation process.     

HPLC determination of antioxidant synergists and ascorbic acid in some fatty pharmaceuticals,cosmetics and food

Summary A specific and sensitive reverse-phase HPLC method for the quantitative determination of ascorbic acid and antioxidant synergists (1-tartaric acid, citric acid, lactic acid as lithium lactate and EDTA) in fatty pharmaceuticals, cosmetics and food has been developed. Two extraction procedures were used; treatment with hot water, and extraction with water from a hexane dilution of the product. No significant differences between the two procedures were found (p<0.05), except for ascorbic acid. Quantitative determinations were performed using a C-18 column and sulfuric acid (pH 1.95) mobile phase. With detection, at 210 nm, lactic acid overlapped with ascorbic acid, but the former could be readily identified by TLC. Ascorbic acid was detected at 254 nm, when lactic acid (as lithium lactate) did not interfere in the analysis. Mean recoveries for tartaric, citric and lactic acids were in the range 96–101%.     

Simple and rugged SPE method for the determination of tetracycline antibiotics in serum by HPLC using a volatile mobile phase

Summary A simple and rugged SPE method for the determination of tetracycline (TC), minocycline (MC) and demeclocycline (DCC) in porcine serum by high performance liquid chromatography (HPLC) was developed. The spiked serum sample was pretreated with 2% phosphoric acid followed by a simple and rugged solid-phase extraction procedure using the Oasis^TM HLB extraction cartridges. High and reproducible recoveries were obtained even though the cartridges were run dry. The extracted sample analytes were injected onto a Waters SymmetryShield^TM RP₈ column. The mobile phase was a simple volatile solution containing 0.1% TFA, 2% methanol and 7% acetonitrile in Water. The antibiotics were detected at 350 nm. The calibration curves were linear from 2.0 to 25.0 μg mL⁻¹ of TC and MC with DCC as the internal standard at a concentration of 25.0 μg mL⁻¹. For six replicate analyses, the average recoveries of TC and MC from porcine serum sample fortified at the level of 2.5 μg mL⁻¹ were 96.1% with 1.3% RSD and 101% with 0.54% RSD; at level of 0.5 μg mL⁻¹ the average recoveries were 88% with 1.6% RSD and 97.8% with 1.4% RSD.     

Simple HPLC method for simultaneous determination of acetaminophen, caffeine and chlorpheniramine maleate in tablet formulations

Summary A simple, rapid and accurate, routine-HPLC method is described for simultaneous determination of acetaminophen, caffeine and chlorpheniramine maleate in a new tablet formulation Chromatographic separation of the three pharmaceuticals was achieved on a Hypersil CN column (150×5.0 mm, 5 μm) using a mobile phase comprising a mixture of acetonitrile, an ion-pair solution and tetrahydrofuran (13:14:87, v/v,pH4.5). The flow-rate was changed from 1.0 mL min⁻¹ (in 0≈7.5 min) to 1.8 mL min⁻¹ (after 3.5 min). was complete in <10 min. The method was validated for system suitability, linearity, accuracy, precision, limits of detection and quantitation, and robustness. Linearity, accuracy and precision were found to be acceptable over the ranges 31.6≈315.8 μg mL⁻¹ for acetaminophen, 9.5≈94.6 μg mL⁻¹ for caffeine and 1.4≈13.8 μg mL⁻¹ for chlorpheniramine maleate.     

Column purification and HPLC determination of teicoplanin and A40926

Summary A simple HPLC method for quali- and quantitative determination of teicoplanin and A40926 in fermentation broths and in biological fluids is described. The sample is purified on affinity chromatography and then analyzed on a reverse phase column using a gradient phosphate buffer-acetonitrile as mobile phase and UV detection at 254 nm. No interference from endogenous sources has been observed. The analytical method, with partial modifications, was also used to isolate on semipreparative scale the single components of these antibiotics, in order to investigate their structure and their biological activity.     

Highly sensitive measurement of mepindolol in biological fluids using HPLC and electrochemical detection

Summary Mepindolol, a β-blocker agent, has an indolic structure which can undergo oxidation. A high-performance liquid chromatographic technique has been used to measure mepindolol in biological fluids using pindolol as the internal standard. The chromatography has been combined with electrochemical detection (coulometric detector). This method allows the determination of very low amounts of mepindolol with good precision and accuracy, the limit of quantification being 0.6 ng · ml⁻¹.     

Comparison of different methods for the determination of several quinolonic and cinolonic antibiotics in trout muscle tissue by HPLC with fluorescence detection

Summary Quinolonic and cinolonic derivatives are mainly used as antibacterials in fish-farms. In this paper we describe a careful revision of the treatment procedures of samples and a prodedure for the determination of residues of these compounds. Because of the complexity and duration of these procedures, several studies have been carried out and these have lead to a simpler and shorter method. Three approaches have been examined: lyophilization followed by extraction with chloroform, solid-liquid extraction with chloroform and solid-liquid extraction with sodium hydroxide solution, followed by liquid-liquid partition in chloroform. Some previous studies into the partition equilibrium are also included. As a result of our studies we propose a procedure with a lower number of steps than those previously described in the literature. This method has been applied to the analysis of nalidixic, 7-hydroxymethylnalidixic and oxolinic acids and cinoxacin in trout muscle. These analysis have been carried out using an HPLC system equipped with a C₁₈ column and fluorimetric detection. The mobile phase was acetonitrile:oxalic acid. The recoveries obtained were: 70–97% for 7-hydroxymethylnalidixic acid, 75–78% for nalidixic acid, 71–95% for oxolinic acid and 72–85% for cinoxacin.     

Comparative Evaluation of CE and HPLC for Determination of Cotinine in Human Urine

Two rapid and popular methods—capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) have been compared for analysis of cotinine in human urine. Cotinine was analyzed in less than 7 min, with detection limits of 5 and 3.2 ng mL⁻¹ for CE and HPLC, respectively. The performance of the methods was evaluated in terms of sensitivity, specificity, precision, accuracy, and limits of detection and quantification. Calibration plots were linear in the range 50–4,000 ng mL⁻¹, at least, and mean recoveries were satisfactory for both techniques. The methods were successfully used for quantification of cotinine in urine.     

A stability-indicating HPLC method for the determination of benzalkonium chloride in 0.5% tramadol ophthalmic solution

Summary A high-performance liquid chromatographic procedure employing ultraviolet detection for the analysis of benzalkonium chloride in 0.5% Tramadol ophthalmic solution is reported. The method requires minimal sample pretreatment and is sensitive, accurate, and reproducible. The peak area versus BAK concentration is linear over the range of 50–150% of its label claim of 0.05 mg/mL. The mean absolute recovery of BAK using the described method is 100.2±1.2%, (mean ±SD, n=10). A stress study with heat, acid, base and UV radiation indicates that the method is stabilityindicating with no interference from drug or degradation products.