Rapid quantification of the delta-opioid receptor selective enkephalin DPDPE in canine cerebrospinal fluid by liquid chromatography--mass spectrometry

An atmospheric pressure ionization liquid chromatographic-mass spectrometric assay was developed and validated for the determination of D-penicillamine(2,5) enkephalin (DPDPE) in cerebrospinal fluid (CSF) from dog. DPDPE and internal standard (D-Ala(2),D-Leu(5) enkephalin=DADLE) were isolated from CSF by reversed-phase C(18) solid-phase extraction with ZipTip micro-cartridges. Aliquots of extracted eluate were injected onto an Agilent Zorbax SB C(18) column (30 x 2.2 mm; 3.5 microm) at a flow-rate of 0.4 ml/min. The isocratic mobile phase of methanol-10 mM ammonium formate (pH 3) (75:25, v/v) was then diverted to waste for 45 s after injection, after which time flow was directed to the single quadrupole mass spectrometer. DPDPE was detected by positive mode selected ion monitoring. Standard curves were linear (r(2)> or =0.991) over the concentration range 1-1000 ng/ml. The efficiency of extraction recovery was greater than 97%, and the intra-assay and inter-assay precisions were within 9% relative standard deviation. DPDPE and the internal standard were stable in the injection solvent at 4 degrees C for at least 48 h. The assay was applied to the pharmacokinetic study of intrathecal DPDPE administration in the dog animal model.     

Liquid chromatography-tandem mass spectrometric determination of a new antibacterial agent (AVE6971) in human white blood cells

An LC-MS/MS assay for the quantitative determination of a new antibacterial agent (AVE6971) has been developed and validated in human white blood cells (WBC). The assay involved a lysing procedure of white blood cells and ultra centrifugation of the extracts. Chromatography was performed on a Supelcosil ABZ+ C(18) (2.1 mm x 50 mm, 5 microm) column using a mobile phase consisting of methanol/acetonitrile/10mM ammonium formate mixture (10:30:60, v/v/v) at a flow rate of 0.2 ml/min. The linearity was within the range of 10-10000 ng/ml of extracts, corresponding to 0.5-500 ng of AVE6971 in WBC pellets tubes. The validated lower limit of quantification was 10 ng/ml. The inter- and intra-run coefficients of variation (CV) for the assay were <12.9% and the accuracy were from -9.0 to -1.2%. AVE6971 was stable in WBC for at least 1 month at -75 degrees C. This assay proved to be suitable for the determination of AVE6971 in WBC from clinical studies.     

Biochemical analysis of a native and proteolytic fragment of a high-molecular-weight thermostable lipase from a mesophilic Bacillus sp.

An extracellular lipase was isolated from the cell-free broth of Bacillus sp. GK 8. The enzyme was purified to 53-fold with a specific activity of 75.7 U mg(-1) of protein and a yield of 31% activity. The apparent molecular mass of the monomeric protein was 108 kDa as estimated by molecular sieving and 112 kDa by SDS-PAGE. The proteolysis of the native molecule yields a low molecular weight component of 11.5 kDa that still retains the active site. It was stable at the pH range of 7.0-10.0 with optimum pH 8.0. The enzyme was stable at 50 degrees C for 1 h with a half life of 2 h, 40 min, and 18 min at 60, 65, and 70 degrees C, respectively. With p-nitrophenyl laurate as substrate the enzyme exhibited a K(m) and V(max) of 3.63 mM and 0.26 microM/min/ml, respectively. Activity was stimulated by Mg(2+) (10 mM), Ba(2+) (10 mM), and SDS (0.1 mM), but inhibited by EDTA (10 mM), phenylmethane sulfonyl fluoride (100 mM), diethylphenylcarbonate (10 mM), and eserine (10 mM). It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C(4) and C(18:1). Thermostability of the proteolytic fragment at 60 degrees C was observed to be 37% of the native protein. The native enzyme was completely stable in ethylene glycol and glycerol (30% v/v each) for 60 min at 65 degrees C.     

Validation and implementation of a liquid chromatography/tandem mass spectrometry assay to quantitate ON 01910.Na, a mitotic progression modulator, in human plasma

A reverse-phase high performance liquid chromatographic method with tandem mass spectrometry (LC-MS/MS) was developed and validated for the quantitation of ON 01910.Na, a novel synthetic benzyl styryl sulfone, in human plasma. The assay involved a simple sample preparation with acetonitrile protein precipitation. ON 01910.Na and the internal standard temazepam were separated on a Waters X-Terra MS C(18) column with mobile phase of acetonitrile containing 0.1% formic acid /10mM ammonium acetate (55:45, v/v) using isocratic flow at 0.2 mL/min for 5 min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Two calibration curves were generated over the range of 10-2000 ng/mL and 100-20000 ng/mL. The lower limit of quantitation (LLOQ) was 10 ng/mL for ON 01910.Na in human plasma. The accuracy and within- and between-day precisions were within the acceptance criteria for bioanalytical assays. ON 01910.Na was found stable in plasma at -70 degrees C for at least 1 year. The method was successfully applied to characterize the plasma concentration-time profiles of ON 01910.Na in the cancer patients in the Phase I study.     

Determination of thalidomide in transport buffer for Caco-2 cell monolayers by high-performance liquid chromatography with ultraviolet detection

We report simple validated HPLC methods for the determination of thalidomide in the transport buffer for the human colonic cell line (Caco-2) cell monolayers. An aliquot of 50 microl of the mixture was injected onto a Spherex C(18) column (150 x 4.6 mm; 5 microm) at a flow-rate of 0.5 ml/min of mobile phase consisting of acetonitrile-10 mM ammonium acetate buffer (24:76, v/v, pH 5.5), and thalidomide was detected by ultraviolet detector at a wavelength of 220 nm. Calibration curves for thalidomide were constructed at the concentration range of 0.025-1.0 and 1.0-50 microM in transport buffer. The validated methods were used to determine the transport of thalidomide by Caco-2 monolayers. The transport across the monolayers from the apical (A) to basolateral (B) side was similar to that from B to A side. The apparent permeability coefficient (P(app)) values of thalidomide at 10-300 microM from the A to B and from B to A side was 2-6 x 10(-5) cm/s, with a marked decrease in P(app) values from A to B side at increased thalidomide concentration. The A to B transport appears to be dependent on temperature and sodium ion. Sodium azide, 2,4-dinitrophenol (both ATP inhibitors), 5-fluorouracil, cytidine and glutamic acid significantly inhibited the transport of thalidomide. These results indicate that the transport of thalidomide by Caco-2 monolayers was rapid, which might involve an energy-dependent mechanism.     

Purification, stability and kinetic properties of highly purified adenosine deaminase from Bacillus cereus NCIB 8122

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from Bacillus cereus NCIB 8122 has been purified to electrophoretic homogeneity by ammonium sulfate precipitation, gel filtration through Sephadex G-100, DEAE-Sephadex A-50 chromatography and ion-exchange HPLC on DEAE-Polyol. The enzyme activity is stabilized (at temperatures from 0 degrees C to 40 degrees C) by 50 mM NH4+ or K+, while it is irreversibly lost in the absence of these or a few other monovalent cations. Glycerol (24% by volume) helps the cation in stabilizing the enzyme activity above 40 degrees C, but also exerts per se a noticeable protecting effect at room temperature. B. cereus adenosine deaminase displays the following properties: Mr on Sephadex G-200, 68,000; Mr in SDS-polyacrylamide gel electrophoresis, 53,700; optimal pH-stability (in the presence of 50 mM KCl) over the range 8-11 at 4 degrees C, and maximal catalytic activity at 30 degrees C between pH 7 and 10; Km for adenosine around 50 microM over the same pH range and Km for 2'-deoxyadenosine around 400 microM.     

Automated liquid-liquid extraction based on 96-well plate format in conjunction with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for the quantitation of methoxsalen in human plasma

A sensitive, specific and high throughput bioanalytical method using automated sample processing via 96-well plate liquid-liquid extraction and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) has been developed for the determination of methoxsalen in human plasma. Plasma samples with ketoconazole as internal standard (IS) were prepared by employing 0.2mL human plasma in ethyl acetate:dichloromethane (80:20, v/v). The chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 column using isocratic mobile phase, consisting of 10mM ammonium formate and acetonitrile (60:40, v/v), at a flow rate of 0.5mL/min. The linear dynamic range was established over the concentration range 1.1-213.1ng/mL for methoxsalen. The method was rugged and rapid with a total run time of 1.5min. It was successfully applied to a pivotal bioequivalence study in 12 healthy human subjects after oral administration of 10mg extended release methoxsalen formulation under fasting condition.     

A liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of valsartan and hydrochlorothiazide in human plasma

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for simultaneous quantification of valsartan and hydrochlorothiazide in human plasma. After a simple protein precipitation using acetonitrile, the analytes were separated on a Zorbax SB-Aq C18 column using acetonitrile-10mM ammonium acetate (60:40, v/v, pH 4.5) as mobile phase at a flow rate of 1.2 mL/min. Valsartan and hydrochlorothiazide were eluted at 2.08 min and 1.50 min, respectively, ionized using ESI source, and then detected by multiple reaction monitoring (MRM) mode. The precursor to product ion transitions of m/z 434.2-350.2 and m/z 295.9-268.9 were used to quantify valsartan and hydrochlorothiazide, respectively. The method was linear in the concentration range of 4-3600 ng/mL for valsartan and 1-900 ng/mL for hydrochlorothiazide. The method was successfully employed in a pharmacokinetic study after an oral administration of a dispersible tablet containing 80 mg valsartan and 12.5 mg hydrochlorothiazide to each of the 20 healthy volunteers.     

Self-association equilibria of Escherichia coli UvrD helicase studied by analytical ultracentrifugation

The Escherichia coli UvrD protein (helicase II) is an SF1 superfamily helicase required for methyl-directed mismatch repair and nucleotide excision repair of DNA. We have characterized quantitatively the self-assembly equilibria of the UvrD protein as a function of [NaCl], [glycerol], and temperature (5-35 degrees C; pH 8.3) using analytical sedimentation velocity and equilibrium techniques, and find that UvrD self-associates into dimeric and tetrameric species over a range of solution conditions (t相似文献   

An ultra performance liquid chromatography-tandem MS assay for tamoxifen metabolites profiling in plasma: first evidence of 4'-hydroxylated metabolites in breast cancer patients

There is increasing evidence that the clinical efficacy of tamoxifen, the first and most widely used targeted therapy for estrogen-sensitive breast cancer, depends on the formation of the active metabolites 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen (endoxifen). Large inter-individual variability in endoxifen plasma concentrations has been observed and related both to genetic and environmental (i.e. drug-induced) factors altering CYP450s metabolizing enzymes activity. In this context, we have developed an ultra performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) requiring 100 μL of plasma for the quantification of tamoxifen and three of its major metabolites in breast cancer patients. Plasma is purified by a combination of protein precipitation, evaporation at room temperature under nitrogen, and reconstitution in methanol/20 mM ammonium formate 1:1 (v/v), adjusted to pH 2.9 with formic acid. Reverse-phase chromatographic separation of tamoxifen, N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen is performed within 13 min using elution with a gradient of 10 mM ammonium formate and acetonitrile, both containing 0.1% formic acid. Analytes quantification, using matrix-matched calibration samples spiked with their respective deuterated internal standards, is performed by electrospray ionization-triple quadrupole mass spectrometry using selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of relative matrix effects variability, as well as tamoxifen and metabolites short-term stability in plasma and whole blood. The method is precise (inter-day CV%: 2.5-7.8%), accurate (-1.4 to +5.8%) and sensitive (lower limits of quantification comprised between 0.4 and 2.0 ng/mL). Application of this method to patients' samples has made possible the identification of two further metabolites, 4'-hydroxy-tamoxifen and 4'-hydroxy-N-desmethyl-tamoxifen, described for the first time in breast cancer patients. This UPLC-MS/MS assay is currently applied for monitoring plasma levels of tamoxifen and its metabolites in breast cancer patients within the frame of a clinical trial aiming to assess the impact of dose increase on tamoxifen and endoxifen exposure.     

Measurement of saturable and non-saturable components of anandamide uptake into P19 embryonic carcinoma cells in the presence of fatty acid-free bovine serum albumin

There is considerable controversy at present concerning the mechanisms responsible for the cellular uptake of anandamide. One particular issue concerns whether fatty acid-free bovine serum albumin should be used in the assays, it having been argued that such a presence effectively prevents the specific uptake of anandamide. In the present study, it has been demonstrated that in the presence of a low (0.1%, w/v) concentration of fatty acid-free bovine serum albumin, a temperature-dependent and saturable (K(m) approximately 1 microM) uptake of anandamide into P19 embryonic carcinoma cells can be demonstrated using an incubation time of 4 min. Under these conditions, the uptake of anandamide at 4 degrees C is low at a substrate concentration of 100 nM. The uptake at 37 degrees C was not significantly reduced following treatment of the cells with either methyl-beta-cyclodextrin (50 microM) or mevinolin (1 microM), but was reduced by the FAAH inhibitor URB597 (1 microM) and inhibited by the transport inhibitor cum FAAH substrate AM404 with an IC(50) value of 12 microM. When a 45 s incubation time was used, the uptake of anandamide was not saturable at 37 degrees C over the concentration range tested (0.1-1 microM). Analysis of the data at 37 degrees C obtained with 45 s, 4 min and 15 min incubation times revealed a very rapid (i.e. complete by 45 s) non-saturable component followed by a slower saturable (K(m) approximately 1 microM) component of the uptake. It is concluded that the presence of a low concentration of fatty acid-free bovine serum albumin at a suitable concentration reduces non-specific binding (and release) of anandamide to cell culture wells, greatly reduces the cellular accumulation seen at 4 degrees C, and allows the visualisation of both non-saturable and saturable components of the uptake to be seen at 37 degrees C.     

Evidence for two genetically distinct DNA primase activities specified by plasmids of the B and I incompatibility groups.

Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50 mumol of methyl viologen per min per mg of protein and 8.2 mumol of coenzyme F420 per min per mg of protein. The apparent Km for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F420, was 10-fold greater (63 microM) than for coenzyme F420 (6 microM). The purified enzyme also reduced flavin mononucleotide (Km = 13 microM) and flavin adenine dinucleotide (Km = 25 microM) with formate, but did not reduce NAD+ or NADP+. The reduction of NADP+ with formate required formate dehydrogenase, coenzyme F420, and coenzyme F420:NADP+ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F420. The optimal reaction rate occurred at 55 degrees C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 microM), azide (Ki = 39 microM), alpha,alpha-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm.     

Determination of MS-275, a novel histone deacetylase inhibitor, in human plasma by liquid chromatography-electrospray mass spectrometry

A rapid method was developed for the quantitative determination of the novel histone deacetylase inhibitor, MS-275, in human plasma. Calibration curves were constructed in the range of 1-100 ng/ml, and were analyzed using a weight factor proportional to the nominal concentration. Sample pretreatment involved a one-step protein precipitation with acetonitrile of 0.1 ml samples. The analysis was performed on a column (75 mm x 4.6 mm i.d.) packed with 3.5 microm Phenyl-SB material, using methanol-10mM ammonium formate (55:45 (v/v)) as the mobile phase. The column effluent was monitored by mass spectrometry with positive electrospray ionization. The values for precision and accuracy were always < or =5.58 and <11.4% relative error, respectively. The method was successfully applied to examine the pharmacokinetics of MS-275 in a cancer patient.     

Development and validation of a highly rapid and sensitive LC-MS/MS method for determination of SZ-685C, an investigational marine anticancer agent, in rat plasma--application to a pharmacokinetic study in rats

A sensitive and rapid method was developed and validated for the quantitative analysis of the novel anticancer agent SZ-685C in rat plasma using high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) in negative ion mode in order to support the following pre-clinical and clinical studies. SZ-685C and the internal standard (IS, emodin) were extracted from rat plasma by a simple liquid-liquid extraction technique using ethyl acetate as extraction solvent. Chromatographic separation was performed on an Elite Hypersil BDS C18 column (100 mm × 2.1 mm i.d., 3 μm). Elution was carried out using methanol/acetonitrile/2mM ammonium formate (pH 4) (80:15:5 (v/v/v)) at a flow-rate of 0.3 mL/min with a run time of 2.5 min. This assay was linear over a concentration range of 50-10,000 ng/mL with a lower limit of quantification of 50 ng/mL. The intra- and inter-batch precision was less than 15% for all quality control samples at concentrations of 100, 1000 and 7500 ng/mL. These results indicate that the method was efficient with a short run time and acceptable accuracy, precision and sensitivity. This method was successfully applied to explore pharmacokinetics of SZ-685C in rats after oral and intravenous administration of this agent. The absolute bioavailability is about 54.8-66.8% and the t(1/2) is 5.7-9.2h, these results provide basic information for further comprehensive pre-clinical research.     

A sensitive LC-MS/MS method for determination of levamisole in human plasma: application to pharmacokinetic study

A sensitive and rapid LC-MS/MS method was developed and validated for the determination of levamisole in human plasma. The assay was based on liquid-liquid extraction of analytes from human plasma with ethyl ether. Chromatographic separation was carried on an Agilent HC-C(8) column (150 mm × 4.6 mm, 5 μm) at 40°C, with a mobile phase consisting of acetonitrile-10 mM ammonium acetate (70:30, v/v), a flow rate of 0.5 mL/min and a total run time of 6 min. Detection and quantification were performed by mass spectrometry in the multiple reaction monitoring mode with positive electrospray ionization m/z at 205.1→178.2 for levamisole, and m/z 296.1→264.1 for mebendazole (internal standard). The assay was linear over a concentration range of 0.1-30 ng/mL with a lower limit of quantification of 0.1 ng/mL. The coefficient of variation of the assay precision was less than 8.5%. The assay was successfully used to analyze human plasma samples in a pharmacokinetic study where levamisole was administered as a liniment.     

Cysteine synthase of an extremely thermophilic bacterium,Thermus thermophilus HB8

O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) was first purified from an extremely thermophilic bacterium, Thermus thermophilus HB8, in order to ascertain that it is responsible for the cysteine synthesis in this organism cultured with either sulfate or methionine given as a sole sulfur source. Polyacrylamide gel electrophoreses both with and without SDS found high purity of the enzyme preparations finally obtained, through ammonium sulfate fractionation, ion exchange chromatography, gel filtration, and hydrophobic chromatography (or affinity chromatography). The enzyme activity formed only one elution curve in each of the four different chromatographies, strongly suggesting the presence of only one enzyme species in this organism. Molecular masses of 34,000 and 68,000 were estimated for dissociated subunit and the native enzyme, respectively, suggesting a homodimeric structure. The enzyme was stable at 70 degrees C at pH 7.8 for 60 min, and more than 90% of the activity was retained after incubation of its solution at 80 degrees C with 10 mm dithiothreitol. The enzyme was also quite stable at pH 8-12 (50 degrees C, 30 min). It had an apparent Km of 4.8 mM for O-acetyl-L-serine (with 1 mM sulfide) and a Vmax of 435 micromol/min/mg of protein. The apparent Km for sulfide was approximately 50 microM (with 20 mM acetylserine), suggesting that the enzyme can react with sulfide liberated very slowly from methionine. The absorption spectrum of the holo-enzyme and inhibition of the activity by carbonyl reagents suggested the presence of pyridoxal 5'-phosphate as a cofactor. The apo-enzyme showed an apparent Km of 29 microM for the cofactor at pH 8. Monoiodoacetic acid (1 mM) almost completely inactivated the enzyme. The meaning of a very high enzyme content in the cell is discussed.     

Arachidonic acid metabolites by cytochrome P-450 dependent monooxygenase pathway in bovine adrenal fasciculata cells

[1-14C]Arachidonic acid was incubated with microsomes of bovine adrenal fasciculata cells in the presence of 1 mM NADPH for 30 min at 37 degrees C. The metabolites were separated and purified by reverse phase high performance liquid chromatography, and identified by gas chromatography-mass spectrometry. Identified metabolites were four dihydroxyeicosatrienoic acids (DHTs) (5,6-, 8,9-, 11,12-, 14,15-DHTs), 20-hydroxyeicosatetraenoic acid and eicosatetradioic acid. The formation of these metabolites was dependent on NADPH and inhibited by SKF-525A. 14,15-DHT was also formed by isolated bovine adrenal fasciculata cells. These results indicate that cytochrome P-450 dependent arachidonate monooxygenase pathway may exist in bovine adrenal fasciculata cells. Addition of the chemically synthesized epoxyeicosatrienoic acids (EETs) to isolated bovine adrenal fasciculata cells stimulated cortisol production. Among four regioisomeric EETs, 14,15-EET was most potent and stimulated steroidogenesis in a dose-related manner over a range of 0.5 to 5.0 microM.     

Development of a liquid chromatography-mass spectrometry method for monitoring the angiotensin-converting enzyme inhibitor lisinopril in serum

In this study, a sensitive, specific, precise and accurate method for lisonopril quantitative determination in human serum was developed and validated. The method comprises lisinopril isolation from serum by means of solid-phase extraction followed by its quantification by liquid chromatography-mass spectrometry. Chromatographic separation was performed at 55 degrees C on Kromasil C(18) 5 micrometer 250x3.2 mm HPLC column with mobile phase composed of 50 mM ammonium formate buffer (pH 3)-acetonirile-methanol (72:7:21, v/v/v). A Finnigan AQA benchtop mass spectrometer with a pneumatically assisted electrospray (ES) interface and a single quadrupole mass filter was used to detect and quantify lisinopril in column effluent. Ion signals were acquired by selected ion monitoring of the protonated lisinopril ion m/z=406.5 (M+1). The detector response was linear with r>0.9993 in the investigated concentration range 6-150 ng/ml. The mean recovery of lisinopril from serum samples was 88%. The limit of quantitation for lisinopril was 6 ng/ml with a signal-to-noise ratio at this concentration level S/N=34.75+/-3.9 (n=4).     

Quantification of total and free mycophenolic acid in human plasma by liquid chromatography with fluorescence detection

A simple high-performance liquid chromatographic (HPLC) method was developed for the assay of total and free mycophenolic acid (MPA) in human plasma. Prior to analysis, total mycophenolic acid was extracted by protein precipitation and free drug was isolated from plasma samples using ultrafiltration. The extracts were injected onto a Kromasil C8 column at 30 degrees C with excitation and emission wavelengths set at 342 and 425 nm, respectively. The mobile phase was consisted of acetonitrile-32 mM glycine buffer, pH 9.2 (20:80, v/v), at a flow rate of 1.0 ml/min. The method was found to be linear over the concentration range investigated, 0.05-40 mg/l for total mycophenolic acid (r>0.999) and 5-1000 microg/l (r>0.99) for free drug. The percentage error of the analytical method was below 10.9%. The intra- and inter-day reproducibility was adequate with the coefficients of variation of 8.28% or below. The run time were 4 and 6 min for free and total MPA, respectively. The method thus can be effectively applied to measure mycophenolic acid concentrations in clinical samples.     

Determination of intralumenal individual bile acids by HPLC with charged aerosol detection

An isocratic HPLC charged aerosol detector (CAD) method was developed, validated, and applied for the determination of individual bile acids in human gastric and duodenal aspirates. The method requires a low volume of aspirates (50-100 microl) and minimal sample pretreatment. A Hypersil BDS RP-C(18) column (250 x 4.6 mm, 5 microm particle size) was equilibrated with a mobile phase composed of methanol-[ammoniun formate 20 mM, formic acid 0.5%, triethylamine 0.2% (pH 3)] 67:33 v/v. Its flow rate was 1 ml/min. The elution times for taurocholate, glycocholate, taurochenodeoxycholate, ursodeoxycholate, glycochenodeoxycholate, cholate, and glycodeoxycholate were approximately 9.9, 16.2, 18.2, 21.3, 31.6, 34.5, and 38.5 min, respectively. Calibration curves in the mobile phase were constructed in the concentration range of 0.5-500 microM. Limits of detection and quantification were in the range of 0.07-0.60 microM and 0.20-1.80 microM, respectively. This method was applied first, in gastric aspirates collected in the fasted state, in which bile acid presence is minimal and, second, in duodenal aspirates collected in the fed state, in which a large number of potentially interfering compounds exists. Intra-day relative standard deviation in fasted gastric aspirates and in fed duodenal aspirates was less than 2.2% and 6.0%, respectively.