Quantitative determination of miconzole in human serum by high-performance liquid chromatography

Summary A high-performance liqid chromatographic method is reported for the measurement of miconazole in systemic, fungal infectious patients. Pharmacokinetic data are presented for a single patient receiving miconazole therapy. Sample preparation involves protein precipitation by acetonitrile (1:1, vol/vol). Analyses are carried out on a reversed-phae chromatographic system using octadecylsilane stationary phase: a mobile phase consisting of 0.05 M acetate buffer (pH 7.4)acetonitrile (20:80, vol/vol) is used to elaute miconazole is quantified on the basis of ultraviolet absorption at 220 nm. The precision of the method ranged from 3.21% at 0.5 mg/L to 0.85% at 2.0 mg/L. The limit of quantification was established as 0.1 mg/L. Interference from other drugs that are co-adimistered such as amphotericin B, 5-fluorocytosine of ketoconazole and most other comonly encountered drugs was not observed.     

高效液相色谱法准确测定血清中的尿酸

建立了准确测定血清中尿酸含量的高效液相色谱方法,血清样品利用乙腈沉淀蛋白,过滤后直接进样测定。所采用的色谱柱为Inertsil ODS-SP(4.6mm i.d.×250 mm,5μm),柱温25℃,流动相体系为10 mmol/L乙酸铵(pH 4.5),流速为1.0 mL/min,紫外检测波长为280 nm。线性范围12.5~150μg/g,回收率为99.79%~100.5%。本文采用乙酸铵缓冲体系,对环境的污染小,同时也为建立液相色谱-质谱法测定尿酸奠定了良好的基础。     

Quantitative determination of celanide by high-performance liquid chromatography

Methods have been developed for the quantitative determination of celanide [lanatoside C] in substances, in solutions for injection, and in tablets by high-performance liquid chromatography, with the aid of which it is possible to determine small amounts of celanide (0.025 mg/ml) with adequate accuracy. The relative error of the determination does not exceed ±4.0%.All-Union Scientific-Research Institute of Physical Culture, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 476–479, July–August, 1983.     

Quantitative determination of the glycosaminoglycan Delta-disaccharide composition of serum, platelets and granulocytes by reversed-phase high-performance liquid chromatography

Seven Delta-disaccharide standards from heparan sulfate/heparin (HS/H) and nine Delta-disaccharide standards from chondroitin/dermatan sulfate (CS/DS) and hyaluronic acid (HA) were derivatized with the fluorophore 2-aminoacridone (AMAC) and separated in two runs each by reversed-phase HPLC with baseline separation and very short run times. This novel method facilitates the separation of the largest number of Delta-disaccharides from both CS/DS/HA and HS/H with one column and buffer system after fluorophore labeling in two runs at present. For the first time nine glycosaminoglycan (GAG) Delta-disaccharides from CS/DS/HA were separated after fluorophore labeling in one run. The limits of quantification (LOQs) were below 0.2 pmol for CS/DS/HA and HS/H Delta-disaccharides. We demonstrated applicability of our method for biological samples. Furthermore, normal ranges of the GAG Delta-disaccharide compositions from platelets and granulocytes were determined for the first time.     

Highly sensitive determination of free fatty acids in human serum by high-performance liquid chromatography with fluorescence detection

Endotoxins from four bacterial species extracted by three different procedures were acid-methanolyzed and the methyl esters of the fatty acids were analyzed by packed-column gas chromatography. There were qualitative and quantitative differences in the fatty acid profiles of the lipopolysaccharides isolated from four Gram-negative bacteria. Our data show considerable lot-to-lot variations in amounts of four methyl esters from the same bacterial serotype extracted by the same procedure and in the same bacterial serotype extracted by different procedures. These results indicate that extraction and perhaps culture conditions, as well as bacterial species, affect the fatty acid composition of endotoxins, hydrolyzed and derivatized by these procedures.     

Quantitative determination by high-performance liquid chromatography of acetylsalicylic acid and related substances in tablets

High-performance liquid chromatography on a Zorbax C8 7-micron column (25 cm X 0.46 cm I.D.) with methanol-water-1 M phosphoric acid (59:36:5) as the mobile phase has been used for the analysis of several naturally aged batches of fourteen brands of acetylsalicyclic acid tablets. The extraction solvent is methanol, containing 2% v/v of formic acid. Salicylic acid is the main impurity. Acetylsalicylsalicylic acid is the second most important impurity, and the corresponding salicylsalicylic acid is rarely present. Buffered or dispersible tablets contain relatively more of the latter two impurities and eventually also the corresponding higher oligomers. Acetylsalicylic anhydride is always a minor impurity. Comparison is made with classical spectrophotometric methods, which are observed to be selective for salicylic acid.     

Quantitative determination of perfluorooctanoic acid ammonium salt in human serum by high-performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

A sensitive, specific, accurate and reproducible analytical method was developed and validated to quantify perfluorooctanoic acid (PFOA) in human serum. After initial extraction with an ion-paring reagent, the procedure for quantifying PFOA is based on high-performance liquid chromatography (HPLC) interfaced to negative ion tandem mass spectrometry, operating in selected ion monitoring mode. The retention times of PFOA and its internal standard (D,L-malic acid) were 5.85 and 1.70 min, respectively. The assay was linear over the range 0-500 ng/mL, with a lower limit of quantification (LOQ) of 25 ng/mL, and with a coefficient of variation (CV) of 7.3%. The lower limit of detection (LOD) was assessed as 10 ng/mL. The overall precision and accuracy were assessed on three different days. The within- and between-day precision was < or =9.7 and 6.8%, respectively, and the accuracy was in the range 96-114%. The mean extracted recovery assessed at three different concentrations (100, 250, and 500 ng/mL) was always more than 85%. With this method no derivatization procedure was needed, thus avoiding possible thermal and chemical decomposition reactions of PFOA. The assay was applied to quantify perfluorooctanoic acid in serum from employees exposed to fluorochemicals commonly used in industrial applications for polymer production. The quantitative results for PFOA blood levels were found to vary between 100 and 982 ng/mL.     

Quantitative monitoring by high-performance liquid chromatography of the dissociation of human serum cholinesterase by limited proteolysis

Proteolytic action on human serum cholinesterase, a tetrameric enzyme, results in a partial disintegration which can be recorded only qualitatively by time-consuming electrophoretic techniques. In this study, a rapid high-performance liquid chromatographic method was used for the separation and determination of the active dissociation products. Separation of the cholinesterase subunits was accomplished by high-performance gel permeation chromatography on a combination of DIOL columns (Zorbax GF 450/GF 250) in 0.2 M phosphate buffer (pH 7.0). Detection and quantification of enzyme activity in the fractionated eluate were carried out using a Flexigem analyser (substrate, butyrylthiocholine). On limited tryptic digestion of partially purified human ChE, up to three peaks of enzyme activity could be identified. Their elution volumes corresponded to apparent molecular masses of 480,000, 270,000 and 120,000, indicating, in addition to the tetrameric holoenzyme, a dimeric and a monomeric form. Quantification of the relative amounts of individual enzyme activity peaks revealed that in the course of degradation, the dimer appeared first, followed by the monomer. This suggests that the first step in the sequence of dissociation is cleavage of the tetramer into a pair of dimers, then further into the monomeric subunit. During the incubation with trypsin, a significant change in the pattern of the different peaks had already occurred when the total enzyme activity was only slightly reduced.     

Separation of carotenol fatty acid esters by high-performance liquid chromatography

Employing isocratic and gradient-elution high-performance liquid chromatography (HPLC) a number of straight-chain fatty acid esters (decanoate, laurate, myristate, palmitate) of violaxanthin, auroxanthin, lutein, zeaxanthin, isozeaxanthin, and beta-cryptoxanthin, prepared by partial synthesis, have been separated on a C18 reversed-phase column. Several chromatographic conditions were developed that separated a mixture of di-fatty acid esters (dimyristate, myristate palmitate mixed ester, dipalmitate) of violaxanthin, auroxanthin, lutein, and zeaxanthin in a single chromatographic run. Hydroxycarotenoids such as lutein, zeaxanthin, and isozeaxanthin that are not easily separated by HPLC on C18 reversed-phase columns, can be readily separated after derivatization with fatty acids and chromatography of their esters. Chromatographic conditions for optimum separation of carotenoids from various classes are discussed.     

Quantitative determination of phospholipids in amniotic fluid by high-performance liquid chromatography

Summary A new quantitative analytical method for the determination of phospholipids in amniotic fluid by high performance liquid chromatography (HPLC) is described. In addition to the main compounds, phosphatidylcholine (lecithin) and sphingomyelin, the so-called minor phospholipids, phosphatidylglycerol, phosphatidylinositol and phosphatidylethanolamine can also be determined. Separation is achieved using a guard-column of Lichrosorb Si 60 and an analytical column of Lichrosorb DIOL. Acetonitrile/water is used as mobile phase at an elevated temperature. By determining the recovery rates, the within-run and the between-run precision, it was shown that sufficient accuracy and precision could be achieved for all the parameters examined. The method is highly sensitive, the detection limit for sphingomyelin is 0.2 μg and 0.1 μg for all the other components. A single determination of 5 phospholipids in an amniotic fluid sample takes about two hours. By performing simultaneous extractions it is possible to analyse 5 samples per day.     

Quantitative fractionation of serum bilirubin species by reversed-phase high-performance liquid chromatography

A rapid high-performance liquid chromatographic method is described for separating and quantifying four bilirubin species present in serum: bilirubin, bilirubin monoglucuronide, bilirubin diglucuronide, and bilialbumin. Sample preparation consists of dilution with ascorbic acid and dimethylsulfoxide and filtration to remove solid materials. The diluted serum was injected directly onto a wide-pore (300 A) reversed-phase column and the bilirubin species eluted with a water-isopropanol gradient. Excellent agreement was found between the total bilirubin concentration measured by the high-performance liquid chromatographic method and a diazotized sulfanilic acid procedure.     

Fluorescence immunological determination of immunoglobulin G in human serum by high-performance liquid gel-permeation chromatography

Summary A high-performance liquid gel-permeation chromatographic method is described for the determination of human serum immunoglobulin G (IgG) by separating the fluorescent immuno complex from the free fluorescence-labeled antibody. Fluorescence-labeled antibody used in this study was fluorescein isothiocyanate (FITC)-labeled Fab fragment goat anti-human IgG (anti-IgG Fab). Immuno complexes and antibody of different molecular sizes can be separated. FITC-labeled anti-IgG Fab was added to the serum and the mixture is passed through the column. An immuno complex separates as well-delineated peak in the column void volume, and was measured by the fluorescence of the column eluate (Ex=490nm, Em=520nm). The total analysis time for a serum sample was approximately 15min. The minimum detection limit was 25 mg/dl. The relative standard deviation was below 2% (peak area). The results of the HPL-GPC analysis correlate well with those obtained by laser nephelometric assay (r=0.992).