The Separation of Conjugated and Unconjugated Bilirubin in Bile by High-Performance Liquid Chromatography

Abstract

A fast and simple method was developed for the separation of unconjugated bilirubin and its mono- and di-glucuronide conjugates from bile by high-performance liquid chromatography (HPLC). Unconjugated bilirubin was separated on a reversed-phase column using acetonitrile-water (70:30 v/v) as the mobile phase, while the conjugates were separated on a μ-Bondapak-carbohydrate column employing acetonitrile-water (90:10 v/v) as the eluent. The application of this method was demonstrated by the analysis of the bile pigments in rat bile.     

Solid-phase extraction and determination of dansyl derivatives of unconjugated and acetylated polyamines by reversed-phase liquid chromatography: improved separation systems for polyamines in cerebrospinal fluid, urine and tissue

A sensitive and simple liquid chromatographic assay with fluorometric detection for unconjugated and acetylated polyamines in biological fluids is described. After precolumn derivatization with dansyl chloride, unconjugated polyamines and acetylated polyamines were extracted by elution from a Bond-Elut C18 column and then separated on a reversed-phase column with gradient elution. The complete analysis of unconjugated putrescine, spermidine, and spermine in either hydrolyzed urine, cerebrospinal fluid or tissue could be accomplished within 20-26 min, while the simultaneous analysis of unconjugated polyamines and monoacetylpolyamines could be completed within 40 min. Unhydrolyzed urine and cerebrospinal fluid required a Bond-Elut cation-exchange clean-up before dansylation. Standard curves for the assay were linear up to 20 nmol/ml, and the within-day and day-to-day coefficients of variation were between 1.1 and 4.6% and between 1.6 and 11.8%, respectively. Results obtained with the method were compared with results obtained with a well established modified amino acid analyzer method for urine, tissue and cerebrospinal fluid samples. The correlation coefficients between these two methods were in the range 0.933-0.996. Detection limits between 50 and 150 fmol were achieved for unconjugated and acetylated polyamines. Of more than twenty drugs and amines tested for possible interference with the assay, only normetanephrine was found to have the same retention time as the internal standard 1,6-diaminohexane.     

High-performance liquid chromatographic separation of bilirubin conjugates: the effects of change in molarity and pH

A fast, sensitive high-performance liquid chromatographic method has been developed for the separation and quantitation of biliary bile pigments; this utilizes a C18 reversed-phase column with two solvents, a buffer and an organic solvent, which were changed in a linear gradient from a polar to a less polar combination. Nine glycosidic conjugates of bilirubin as well as unconjugated bilirubin and a suitable internal standard, unconjugated mesobilirubin IX alpha, were all separated to baseline by gradient elution; the species eluted in a polar to less polar fashion. Increasing the molarity of the solvent decreased the binding of non-glucuronide pigments to the column, with a decrease in their retention times, whereas for bilirubin monoglucuronide they increased. Decrease in pH, similarly, preferentially increased bilirubin monoglucuronide retention times.     

Chromatographic analysis and structure determination of biliverdins and bilirubins

Recent applications of thin-layer chromatographic (TLC) and high-performance liquid chromatographic (HPLC) procedures has revealed an unexpected wide variety of naturally occurring unconjugated and conjugated bilirubins. Biliverdins seems to occur only in unconjugated forms, mainly as the IX alpha isomer. Several synthetic biliverdins and bilirubins present interesting models for biochemical and metabolic studies. Owing to recent recognition of the astounding heterogeneity of natural bilirubins and to the various artifactual changes that bile pigments can undergo, considerable confusion has existed, and still exists, with regard to the nomenclature of the bile pigments and their derivatives. To set a background for further discussion, the present review starts with a brief discussion of nomenclature and of the various characteristic forms of lability of the bile pigments. TLC and HPLC procedures for preparation and analysis of unconjugated biliverdins and bilirubins and their methyl ester and sugar ester conjugates, as well as procedures for analysis of bilirubin-protein conjugates, are then discussed. Since, in view of the lability and pronounced heterogeneity of bile pigments, it is important to assess the composition and nature of chromatographically isolated pigments, the review is concluded by a brief evaluation of various structural tests.     

Novel method for high-performance liquid chromatography of azo derivatives of conjugated and unconjugated bilirubin

A method for the separation and quantitation of ethyl anthranilate or p-iodoaniline azo derivatives of bile pigments was developed using reversed-phase high-performance liquid chromatography. A convenient separation was achieved in 15 min, permitting the quantitation of the unconjugated azo-dipyrrole (alpha o) and its glucuronide (delta), xyloside (alpha 2) and glucoside (alpha 3) conjugates. The pathological beta- and gamma-azo pigments, derived from bilirubin glucuronide isomers that occur in cholestatic bile or plasma, are also detected in this system. The results of this method as applied to bile from 25 healthy dogs were in excellent agreement with the values obtained by reversed-phase chromatography of bilirubin and its mono- and dimethyl esters produced from the corresponding conjugates by alkaline methanolysis. This system permits the sensitive and convenient determination of bilirubin and its conjugation pattern in biological fluids.     

Fully automated analytical system using liquid-solid extraction and liquid chromatography for the determination of CGP 6140 in plasma

Liquid-solid extraction on disposable extraction columns (DECs) and liquid chromatography can be combined in a completely automated analyser. The Gilson ASPEC system was used to develop a procedure for the determination of CGP 6140 in plasma. Both sample preparation via C8 Bond-Elut DECs and injection were fully automatic. The fully automated system prepared the samples by performing the same operations as for a manual procedure. The DEC was first wetted with methanol, then with water. A 400-microliters volume of plasma and 40 microliters of the internal standard solution, diluted with 1 ml of water, were applied to the DEC, rinsed with 10(-2) mol/l dipotassium hydrogenphosphate and eluted from the DEC with 300 microliters of acetonitrile-methanol (50:50, v/v). The eluting strength of the eluate was reduced by dispensing 1 ml of water into each vial prior to direct injection into a Spherisorb ODS column via a 1-ml loop. This allowed the reconcentration of the extracted compounds on the top of the column, as they were injected in a large volume of solvent of lower eluting strength than the mobile phase [acetonitrile-methanol-4 x 10(-3) mol/l ammonia solution (54.5:5:40.5, v/v/v)]. Reproducibility results are presented.     

HPLC-fluorescence determination of equilin and equilenin in postmenopausal women's urine

A high-performance liquid chromatographic (HPLC) method with fluorescence detection (lambda(ex) = 280 nm; lambda(em) = 410 and 312 nm) in combination with a post-column on-line photochemical derivatization is described for the determination of equilin and equilenin in urine from normal postmenopausal women after therapy with conjugated oestrogens. The column effluents were subjected on-line to UV irradiation (254 nm) and the photo-induced modifications were useful for the identification of the analytes. The conjugated (sulphate and glucuronide) forms were analysed after enzymatic or chemical hydrolysis and extracted with chloroform. Solid-phase extraction using strong anion-exchange sorbent was applied to the analysis of unconjugated oestrogen fraction to obtain a practical and reliable sample clean-up. The HPLC separations were achieved using ODS columns with a mobile phase consisting of 0.05 M triethylamine phosphate buffer (pH 4.0)-acetonitrile (64:36, v/v) at a flow rate of 1.0 mL/min. The method was accurate and reproducible; for the equilin and equilenin separation isocratic conditions were satisfactory, allowing a sensitive detection in urine samples with a detection limit of about 50 fmol for equilin (lambda(ex) = 280 nm; lambda(em) = 312 nm, after photoderivatization) and 10 fmol for equilenin (lambda(ex) = 280 nm; lambda(em) = 410 nm).     

Importance of Bile Composition for Diagnosis of Biliary Obstructions

Determination of the cause of a biliary obstruction is often inconclusive from serum analysis alone without further clinical tests. To this end, serum markers as well as the composition of bile of 74 patients with biliary obstructions were determined to improve the diagnoses. The samples were collected from the patients during an endoscopic retrograde cholangiopancreatography (ERCP). The concentration of eight bile salts, specifically sodium cholate, sodium glycocholate, sodium taurocholate, sodium glycodeoxycholate, sodium chenodeoxycholate, sodium glycochenodeoxycholate, sodium taurodeoxycholate, and sodium taurochenodeoxycholate as well as bile cholesterol were determined by HPLC-MS. Serum alanine aminotransferase (ALT), aspartate transaminase (AST), and bilirubin were measured before the ERCP. The aim was to determine a diagnostic factor and gain insights into the influence of serum bilirubin as well as bile salts on diseases. Ratios of conjugated/unconjugated, primary/secondary, and taurine/glycine conjugated bile salts were determined to facilitate the comparison to literature data. Receiver operating characteristic (ROC) curves were determined, and the cut-off values were calculated by determining the point closest to (0,1). It was found that serum bilirubin was a good indicator of the type of biliary obstruction; it was able to differentiate between benign obstructions such as choledocholithiasis (at the concentration of >11 µmol/L) and malignant changes such as pancreatic neoplasms or cholangiocarcinoma (at the concentration of >59 µmol/L). In addition, it was shown that conjugated/unconjugated bile salts confirm the presence of an obstruction. With lower levels of conjugated/unconjugated bile salts the possibility for inflammation and, thus, neoplasms increase.     

Assay of cholesterol 7 alpha-hydroxylase utilizing a silica cartridge column and 5 alpha-cholestane-3 beta, 7 beta-diol as an internal standard

A simplified and accurate assay of cholesterol 7 alpha-hydroxylase activity using 5 alpha-cholestane-3 beta,7 beta-diol as an internal standard is described. Endogenous microsomal cholesterol was used as the substrate. Following incubation and addition of the internal standard, lipids extracted from the incubation mixture were applied to Bond-Elut silica cartridge columns. 7 alpha-Hydroxycholesterol, 7 beta-hydroxycholesterol, 7-ketocholesterol and the internal standard were quantitatively recovered by eluting the column with 6 ml of benzene-ethyl acetate (2:3, v/v) after removal of cholesterol with 6 ml of benzene-ethyl acetate (9:1, v/v). After trimethylsilylation, the mass of 7 alpha-hydroxycholesterol was determined by capillary gas chromatography with selected-ion monitoring. The method permits a faster, easier and more sensitive determination of the activity of cholesterol 7 alpha-hydroxylase in small samples.     

Application of dansyl derivatization to the high pressure liquid chromatographic identification of equine estrogens

The HPLC qualitative analysis of conjugated estrogens is accomplished by a two-step procedure involving the formation of the corresponding dansyl derivatives. The first step involves the acid hydrolysis of the conjugated estrogens, followed by dansyl derivatization and HPLC separation of these derivatives on a liChrosorb Si-60 column with 50% (v/v) chloroform-n-heptane as the mobile phase. All of the dansyl estrogens are well separated except for the 17-keto estrogens, estrone, equilin, and equilenin. The second step, designed to detect the three 17-keto estrogens, begins with the selective sodium borohydride reduction of the conjugated 17-keto estrogens to the corresponding 17-hydroxyl compounds (the beta-epimer being formed in vast predominance over the alpha-epimer), followed by acid hydrolysis, dansyl derivatization, and HPLC separation of the derivatives as in the first step. Detection of the 17-keto estrogens is possible by determining differences in peak heights between the chromatograms of the first and second analyses. The The proposed method is sensitive, the dansyl derivatives stable, and nine different estrogens can be readily identified.     

High-performance liquid chromatographic analysis of amphotericin B in plasma, blood, urine and tissues for pharmacokinetic and tissue distribution studies.

A sensitive and reproducible high-performance liquid chromatographic method was developed to assay ampherotericin B in plasma, blood, urine and various tissue samples. Amphotericin B was isolated from each sample matrix by solid-phase extraction (Bond-Elut). The eluate from Bond-Elut containing amphotericin B was injected onto a reversed-phase C18 column (Waters, mu Bondpak, 10 microns, 300 mm x 3.9 mm I.D.) with a mobile phase of 45% acetonitrile in 2.5 mM Na2EDTA at 1 ml/min. Detection of amphotericin B was by ultraviolet absorption at 382 nm. Blood and tissues were homogenized and extracted with methanol prior to Bond-Elut extraction. The extraction efficiencies of amphotericin B from plasma, blood and tissues were approximately 90, 70 and 75%, respectively. The sensitivity of the assay was less than or equal to 5 ng/ml for plasma, less than or equal to 25 ng/ml for blood, 2.5 ng/ml for urine and 50 ng/g for tissues. The linearity of the assay method was up to 2.5 micrograms/ml for plasma, 5 micrograms/ml for blood, 500 ng/ml for urine and 500 micrograms/g for tissues. The assay was reproducible with an intra-day coefficient of variation (C.V., n = 3) of less than 5% in general for plasma, blood and tissues. The inter-day C.V. of the assay was less than 5% for plasma (n = 5), less than 10% for blood (n = 4) and less than 5% for tissues (n = 3). The overall variability in the urine assay was generally less than 10%. This method has demonstrated significant improvement in the sensitivity and reproducibility in assaying amphotericin B in plasma and especially in blood, urine and tissues. We have employed this assay to compare the pharmacokinetic and tissue distribution profiles of amphotericin B in rats and dogs following administration of Fungizone and ABCD (amphotericin B-cholesteryl sulfate colloidal dispersion), a lipid-based dosage form. In addition, the assay method for plasma and urine samples can also be applied to pharmacokinetics studies of amphotericin B in man.     

Determination of 17-oxosteroid glucuronides and sulphates in urine by liquid chromatography using 2,4-dinitrophenylhydrazine as a prelabelling reagent for spectrophotometric detection

A direct, convenient method using high-performance liquid chromatography with spectrophotometric detection has been developed for the determination of conjugated 17-oxosteroids in urine without hydrolysis. Conjugated 17-oxosteroids are extracted with a Sep-Pak C18 cartridge, prelabelled with 2,4-dinitrophenylhydrazine in trichloroacetic acid-benzene solution and then separated by high-performance liquid chromatography on a reversed-phase C18 column using a mobile phase of 70% methanol in a buffer consisting of 50 mM sodium acetate in 2% (v/v) acetic acid. The eluate is monitored by a spectrophotometer at 380 nm and a linear response was found for absorbance readings (peak heights) from amounts of various conjugated oxosteroids between 25 and 250 ng. The method provides a sensitive, reliable technique for the analysis of urinary 17-oxosteroid conjugates.     

Capillary electrophoresis for determination of free and albumin-bound bilirubin and the investigation of drug interaction with bilirubin-bound albumin

Capillary electrophoresis (CE) is a promising technique for assessment of free bilirubin and its interaction with albumin, as it requires only a small sample volume and provides a rapid and efficient separation of free bilirubin from its albumin-bound complex in a one-phase system. In order to maintain the equilibrium without dissociation of bilirubin from the albumin/bilirubin complex as in real clinical conditions, the coupling of CE with frontal analysis (FA) was investigated. A very large sample plug was introduced hydrodynamically into the capillary (36 cm length, 50 microm inner diameter) at 15 psi x s to develop the frontal conditions during CE separation. The working conditions for CE/FA separation of bilirubin and albumin were optimized as follows: +20 kV; running buffer, 10 mmol/L phosphate and 1 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 7.4. The working range for bilirubin was found to vary from 5 to 206 micromol/L; precision with relative standard deviation (RSD) <2.0% for n = 3 and detection limit (signal to noise, S/N = 2) was 2 micromol/L. The residual binding capacity of a simulated cord blood serum for bilirubin was 26 mg/100 mL at pH 7.4. Bilirubin was shown to be displaced from albumin when aspirin was added. The free bilirubin concentration was found to increase to clinical significant concentrations from 11.9 to 21.1% when increasing aspirin was added in the range of 5-50 mg/100 mL, respectively. Thus, the investigation of aspirin displacement of bilirubin from albumin is clinically important and the CE/ FA method is a suitable procedure for this purpose.     

胆红素—白蛋白复合物的化学平衡离解常数及结合自由能的测定

胆红素与血清白蛋白结合能形成一种水溶性的复合物［‘，’j，清除血液中过量的胆红素是人工肝辅助装置研究的热门课题［‘1．血液灌流所用材料必须具备与白蛋白竞争吸附胆红素的能力．而关于胆红素与白蛋白结合的平衡常数的测定尚未见报道．本文利用在不同白蛋白浓度和不同胆红素浓度的溶液中，CHP树脂对胆红素的吸附数据，求得胆红素一白蛋白复合物的化学平衡离解常数及结合自由能，方法简便易行．1实验部分1．1试剂及仪器壳聚糖（南开大学高分子所自制）；戊二醛（天津津宁精细化工厂），巳R．级；Span80（天津化学试剂三厂），C．P；…     

Development of a sensitive method for the determination of ganciclovir by reversed-phase high-performance liquid chromatography

Ganciclovir is a nucleoside analogue widely used in the treatment of cytomegalovirus infections, which affects mainly immunocompromised patients. Recently, new pharmaceutical dosage forms based on the use of albumin nanoparticles have been developed for improving the efficacy of this drug. The aim of this study was to develop an analytical HPLC method for the determination of ganciclovir in both pharmaceuticals (i.e. albumin nanoparticles) and biological medium samples. The chromatography was performed on a reversed-phase encapped column (LiChrospher Select B C₈) with a mobile phase consisting of acetonitrile in 0.05 M ammonium acetate (pH 6.5; 2: 98, v/v). Acyclovir was used as internal standard and the detection wavelength was 254 nm. The limit of quantitation of ganciclovir was 50 ng/ml and the average recoveries over a concentration range of 0.05–10 μg/ml ranged from 98 to 102%. Precision did not exceed 5%. In summary, this assay is a selective, sensitive and reproducible method for the determination of the ganciclovir in albumin nanoparticles. It can be successfully applied to the estimation of the ganciclovir uptake by cultured human corneal fibroblasts.     

Determination of direct-bilirubin by a fluorimetric-enzymatic method based on bilirubin oxidase

A method for the determination of direct bilirubin by reaction with bilirubin oxidase (BOx) is reported. The procedure is based on the changes in fluorescence which take place during the enzymatic reaction of BOx with any of the three forms of bilirubin (free, conjugated and with albumin) when the solution is excited at 240 nm and the emission is measured at 440 nm. The change in fluorescence was studied thoroughly. It seems mainly due to the fluorescence of one of the reaction products. A theoretical study was carried out to relate the changes in fluorescence observed to the species taking part in the reaction and to establish some of the enzymatic reaction constants. The optimum reaction conditions were studied for each of the three types of bilirubin together with their analytical characteristics (linear range and precision). Selective determination of direct bilirubin was carried out for various synthetic samples with good results. A linear response up to 7 mg L^–1 of direct bilirubin was obtained. Using optimum conditions, the precision for free and conjugated bilirubin was 3.4% (n = 5) and 3.0% (n = 5), respectively. Received: 24 January 2000 / Revised: 9 May 2000 / Accepted: 15 May 2000     

High-performance liquid chromatographic determination of carbamazepine and carbamazepine 10,11-epoxide in plasma and saliva following solid-phase sample extraction

A rapid, sensitive and simple to operate high-performance liquid chromatographic method for the simultaneous determination of carbamazepine (CBZ) and carbamazepine 10,11-epoxide (CBZ-EP) in plasma and saliva is described. The drug and its metabolite are extracted from both plasma and saliva using commercially available reversed-phase octadecylsilane bonded silica columns (Bond-Elut C18, 2.8 ml capacity). Separation of CBZ and CBZ-EP was achieved by reversed-phase chromatography, using a mobile phase consisting of acetonitrile-methanol-water (19:37:44) at a flow-rate of 1.8 ml/min in conjunction with a Nova-Pak C18 column. The analytical column, in Radial-Pak cartridge form, was used in combination with a Z-module RCSS and protected by a Guard-Pak precolumn module containing a Guard-Pak mu Bondapak C18 insert. Using ultraviolet detection at 214 nm, levels in the region of 50-100 ng/ml for CBZ and CBZ-EP can be measured with only 250 and 500 microliters of plasma and saliva, respectively. The method, which has been used to determine steady-state concentrations of the drug and its metabolite in paediatric patients receiving CBZ monotherapy, is also suitable for pharmacokinetic studies.     

Determination of direct-bilirubin by a fluorimetric-enzymatic method based on bilirubin oxidase

A method for the determination of direct bilirubin by reaction with bilirubin oxidase (BOx) is reported. The procedure is based on the changes in fluorescence which take place during the enzymatic reaction of BOx with any of the three forms of bilirubin (free, conjugated and with albumin) when the solution is excited at 240 nm and the emission is measured at 440 nm. The change in fluorescence was studied thoroughly. It seems mainly due to the fluorescence of one of the reaction products. A theoretical study was carried out to relate the changes in fluorescence observed to the species taking part in the reaction and to establish some of the enzymatic reaction constants. The optimum reaction conditions were studied for each of the three types of bilirubin together with their analytical characteristics (linear range and precision). Selective determination of direct bilirubin was carried out for various synthetic samples with good results. A linear response up to 7 mg L(-1) of direct bilirubin was obtained. Using optimum conditions, the precision for free and conjugated bilirubin was 3.4% (n = 5) and 3.0% (n = 5), respectively.     

液相色谱/四极杆-飞行时间质谱法筛查奶酪中29种禁用和限用合成色素

建立了奶酪样品中29种禁用和限用合成色素的液相色谱/四极杆-飞行时间质谱(LC/Q-TOF MS)筛查方法。样品经正己烷-水(3:1, v/v)振荡提取,得到正己烷层、水层和残渣3部分。正己烷层经旋转蒸发浓缩后,用乙酸乙酯-环己烷(1:1, v/v)溶解,经过凝胶渗透色谱(GPC)净化去除油脂。水层经乙腈振荡提取,得到乙腈-水提取液。残渣经氨水-甲醇(1:99, v/v)溶液振荡提取,得到氨水-甲醇提取液。乙腈-水提取液和氨水-甲醇提取液不需净化直接分析。结果表明: 29种不同极性范围的合成色素化合物分别得到了有效的提取,提取回收率为70%～95%。而Q-TOF MS提供的精确质量数定性功能可以将不同种类的合成色素化合物筛查出来,各化合物与精确质量质谱库中化合物的匹配度为59.66～99.47。通过Target MS/MS扫描方式进行定量,得到8种苏丹类化合物的方法检出限为0.4～2.5 μg/kg, 21种水溶性合成色素及染料化合物的检出限为20～80 μg/kg。该方法对禁用和限用合成色素的筛查范围广泛,对含有蛋白质、脂肪等基质的食品具有较好的适用性。     

正交试验法优化制备用于小分子物质分离的聚(甲基丙烯酸缩水甘油酯-二乙烯基苯)型整体柱

以甲基丙烯酸缩水甘油酯(GMA)为单体,二乙烯基苯(DVB)为交联剂,以环己醇和正十二醇为致孔剂,过氧化苯甲酰(BPO)为引发剂,直接以50 mm×4.6 mm色谱柱为模具,通过原位聚合制备聚(GMA-DVB)型整体柱。以GMA和DVB的体积比、环己醇和十二醇的体积比以及BPO占聚合物的质量分数为三因素,以分离苯和乙苯等小分子物质时的半峰宽分离度(R1/2)为考察指标,进行三因素三水平的正交试验,通过测定整体柱的比表面积、孔径和孔容分布对其进行表征。结果表明,制备整体柱的最优配方为V(GMA): V(DVB): V(环己醇): V(正十二醇)=0.825:0.825:1.32:2.03, BPO的质量分数为0.7%。应用所制备的整体柱分离苯和乙苯等小分子物质,理论塔板数达到37000塔板/m, R1/2值达到7.14,完全达到基线分离,分离时间小于10 min。该方法制备整体柱的重复性好,柱效较高,基本满足商品化要求。