High-performance liquid chromatographic separation of a complex mixture of diuretics using a micellar mobile phase of sodium dodecyl sulphate: Application to human urine samples

A systematic optimization of the HPLC separation of a complex mixture containing 19 diuretics by micellar liquid chromatography using sodium dodecyl sulphate (SDS), a Hypersil (150 mm×3.0 mm I.D., 5 μm) C₁₈ column, a flow-rate of 0.5 ml min⁻¹ and UV absorbance detection has been carried out. Several mobile phases consisting of SDS and organic modifiers such as acetonitrile, tetrahydrofuran, propanol, butanol or pentanol, and the pH adjusted to 3.2, were tested. The effect of the organic modifier and SDS concentration on the retention behavior and separation of the diuretics was investigated. A mobile phase containing 40 mM SDS and 4% tetrahydrofuran was finally selected. Under these conditions, 14 out of 19 diuretics were separated in about 31 min. A bivariant optimization method for the mobile phase SDS–tetrahydrofuran corroborated the above results. The effect of temperature on the retention was also studied, and 50°C was selected. The optimized method was applied to human urine samples of subjects administered Diurex^® (tablets containing 20 mg of the active ingredient xipamide) without sample preparation.     

Quantitation of hepcidin in human urine by liquid chromatography-mass spectrometry

Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Serum and urine levels are increased in inflammation and suppressed in hemochromatosis, and they may have diagnostic importance. This study describes the development and validation of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. A stable, isotopically labeled internal standard, [¹⁵N,¹³C₂]Gly12,20-hepcidin, was synthesized and a standard quantity was added to urine samples. Extraction was performed using weak cation exchange magnetic nanoparticles. An ion trap mass spectrometer was used to quantify hepcidin in the samples. The hepcidin assay was validated, and good recovery of hepcidin was obtained. The assay is accurate and precise. Urinary hepcidin levels of 3 to 9 nmol/mmol creatinine⁻¹ were found in healthy controls, with reduced levels in hemochromatosis (P < 0.00006) and elevated levels in inflammation (P < 0.00035). In sickle cell disease, a wide range was found, with the mean value not differing significantly from controls (P < 0.26). In summary, a validated method has been developed for the quantitation of hepcidin using a stable, isotopically labeled internal standard and applied to determine the concentrations of hepcidin in the low nanomolar range in urine samples from patients and controls.     

Determination of phloroglucinol in human plasma by high-performance liquid chromatography-mass spectrometry

A sensitive and selective liquid chromatographic method coupled with mass spectrometry (LC-MS) was developed for the quantification of phloroglucinol in human plasma. Resorcinol was used as internal standard, with plasma samples extracted using ethyl acetate. A centrifuged upper layer was then evaporated and reconstituted with mobile phase. The reconstituted samples were injected into a C(18) XTerra MS column (2.1 x 100 mm) with 3.5-microm particle size. The analytical column lasted for at least 500 injections. The mobile phase was 15% acetonitrile (pH 3.0), with flow-rate at 200 microl/min. The mass spectrometer was operated in negative ion mode with selective ion monitoring (SIM). Phloroglucinol was detected without severe interferences from plasma matrix when used negative ion mode. Phloroglucinol produced a parent molecule ([M-H](-)) at m/z 125 in negative ion mode. Detection of phloroglucinol in human plasma was accurate and precise, with quantification limit at 5 ng/ml. This method has been successfully applied to a study of phloroglucinol in human specimens.     

Comparison of gas chromatography-mass spectrometry and high-performance liquid chromatography with coulometric electrode array detection for determination of alkylresorcinol metabolites in human urine

Alkylresorcinols (AR) are amphiphilic compounds present at high concentrations in the outer parts of wheat and rye kernels. Due to their specificity to whole grain and bran products of these cereals, AR and their metabolites have been proposed as biomarkers for intake of such foods. Two alkylresorcinol metabolites, 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA), have previously been quantified in human urine using two different methodologies: high-performance liquid chromatography coupled to a coulometric electrode array detector (HPLC-CEAD) and gas chromatography in combination with mass spectrometry (GC-MS). In this study, these two methodologies were compared by analysing 114 urine samples from free-living Swedish subjects consuming their habitual diet. Data were evaluated by graphical investigation of difference-plots and statistical inference of agreement was assessed by weighted Deming regression analysis. The median DHBA concentrations were 11 μM (GC-MS) and 13 μM (HPLC-CEAD), respectively. Both difference-plot and regression analysis showed a small but statistically significant additive bias, with HPLC-CEAD resulting in a slightly higher DHBA concentration than GC-MS. The median concentration of DHPPA was 18 μM for both methods. Examination of the difference-plot of DHPPA did not indicate any systematic difference between the methods, but regression analysis showed small but statistically significant constant and proportional biases. The conclusion was that the two methodologies are equally suitable for analysis of alkylresorcinol metabolites in human urine and that any small systematic differences observed are most likely of limited practical importance.     

Determination of fexofenadine in human plasma and urine by liquid chromatography-mass spectrometry

A sensitive method was developed to determine fexofenadine in human plasma and urine by HPLC-electrospray mass spectrometry with MDL 026042 as internal standard. Extraction was carried out on C18 solid-phase extraction cartridges. The mobile phases used for HPLC were: (A) 12 mM ammonium acetate in water and (B) acetonitrile. Chromatographic separation was achieved on a LUNA CN column (10 cm x 2.0 mm I.D., particle size 3 microm) using a linear gradient from 40% B to 60% B in 10 min. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH+ ions, m/z 502.3 for fexofenadine and m/z 530.3 for the internal standard. The limit of quantification achieved with this method was 0.5 ng/ml in plasma and 1.0 ng in 50 microl of urine. The method described was successfully applied to the determination of fexofenadine in human plasma and urine in pharmacokinetic studies.     

Detection of hydroxyethylstarch (HES) in human urine by liquid chromatography-mass spectrometry

The objective of this study was to establish the possibility of using liquid chromatography coupled to mass spectrometry for the detection of hydroxyethylstarch (a corn starch derived product) in urine as an alternative to the current time consuming GC-MS methods. Analyses were performed using an ion trap instrument after acidic hydrolysis. Ionization was carried out using atmospheric pressure chemical ionisation (APCI) operated in negative ionization mode and detection was performed using MS(2). The results indicate that the developed method can successfully be applied as a fast and reliable method for the detection and identification of hydroxyethylstarch.     

Application of liquid chromatography-mass spectrometry to the quantification of bisphenol A in human semen

The potential risks to human health and reproduction from the xenoestrogen bisphenol A (BPA) have not been well established. This is due in part to the absence of accurate analytical methods to quantify BPA in biological samples. In this study we establish an accurate, sensitive and selective analytical method for the quantification of BPA in human semen. To quantify BPA we compared the techniques of liquid chromatography-mass spectrometry (LC-MS) and enzyme-linked immunosorbent assay (ELISA). In addition we have taken steps to eliminate BPA contamination during sample extraction and preparation. Results show that the ELISA method gives an over-estimate of BPA concentration, which may be due, at least in part, to non-specific interactions with the BPA-antibodies. LC-MS gave much more accurate results and proved to be more sensitive with a detection limit of 0.5 ng ml(-1) compared to 2.0 ng ml(-1) by ELISA.     

Picomolar concentrations of morphine in human urine determined by dansyl derivatization and liquid chromatography-mass spectrometry

Morphine is present in varying amounts as an endogenous product in human urine. Derivatization of morphine contained in urine with dansyl chloride yields a known product, which can be quantified by liquid chromatography mass spectrometry with high selectivity and sensitivity. Urine samples of 51 healthy individuals were spiked with stable-isotope labeled morphine, hydrolyzed and subjected to solid phase extraction followed by derivatization of morphine with dansyl chloride. The dansyl derivatives of naturally occurring morphine and deuterated internal standard were then detected by liquid chromatography-triple quadrupole mass spectrometry. Using the [N-CD(3)]-labeled internal standard and solid-phase extraction, a limit of detection of 35 fmol/ml (10 pg/ml) and a limit of quantification of 87.5 fmol/ml (25 pg/ml) was determined for morphine in human urine. This new LC-MS/MS method allowed the detection of endogenous morphine in human urine of 51 volunteers with an average value of 156.4 fmol/ml (44.7 ng/ml).     

Application of a validated high-performance liquid chromatography-mass spectrometry assay to the analysis of m- and p-hydroxybenzoylecgonine in meconium

A high-performance liquid chromatography (HPLC)-mass spectrometry (MS) assay, already validated for opiates and cocaine in meconium, has been re-applied for determination of m- and p-hydroxybenzoylecgonine, using nalorphine as the internal standard. Methodology included an initial extraction from the matrix by methanol and then a solid-phase extraction (SPE). A reversed-phase chromatography was used with a gradient of 1% acetic acid-acetonitrile coupled to atmospheric pressure ionization electrospray-mass spectrometry single ion monitoring mode. This method, validated in the range 0.005-1.00 microg analytes/g meconium, proved useful to identify and quantify these two metabolites in meconium samples, already tested for the presence of cocaine, benzoylecgonine and cocaethylene. A positivity of range of concentrations varied between 0.007 and 0.338 microg/g, confirming the importance of these two hydroxylated derivatives to monitor fetal exposure to cocaine.     

Application of high-performance liquid chromatography with a chemiluminescence detection system to determine catecholamines in urine

Chemiluminescence generated with the reaction of bis(2,4,6-trichlorophenyl)oxalate and hydrogen peroxide was applied to a detection system for high-performance liquid chromatography to determine fluorescamine-labeled catecholamines. The sensitivity of the chemiluminescence detection system with 25 fmol of detection limit was approximately 20 times higher than that of a conventional fluorescence detection system. Norepinephrine and dopamine in human urine were determined by the use of the new high-performance liquid chromatography detection system with the coefficient of variation of less than 4.0%. Good correlations (r = 0.998 for norepinephrine and r = 0.999 for dopamine) were obtained between the values by the present method and the conventional method.     

Determination of 5-methyltetrahydrofolic acid in human serum by stable-isotope dilution high-performance liquid chromatography-mass spectrometry.

The need for specific and sensitive methods for the determination of distinct serum folates is of high priority in clinical research settings. A stable-isotope liquid chromatography-mass spectrometry (LC/ESI-MS) assay was developed for the quantitative determination of the monoglutamyl form of 5-methyltetrahydrofolic acid (5-MTHFA) in human serum. Serum samples (0.5 ml) were amended with the internal standard, [5-13C5]MTHFA that had been labeled on the glutamic acid portion of the molecule and allowed to equilibrate. The analyte was trapped onto a solid-phase cartridge and then eluted with the HPLC mobile phase. Forty microliters was taken for LC/ESI-MS analysis using electrospray ionization operated in the positive ion mode. Using the standard method of addition of 5-MTHFA to serum, a linear dilution curve (y = 12.777x - 1.404; range 0.94-97 ng x ml(-1)) was constructed. The precision of the method was 5.3% (CV) based on the analysis of four sample replicates. The mass spectrum produced upon collision induced dissociation of the analyte in serum was used to confirm the identity of the 5-MTHFA. The method was applied to the analysis of a set of serum samples that contained standardized concentrations of 5-MTHFA. The determinations of 5-MTHFA in these samples using the LC/ESI-MS procedure were found to be in good agreement with other folate methods. A highly accurate and specific method for the analysis of 5-MTHFA in serum has been developed utilizing stable isotope dilution mass spectrometry.     

Determination of carboplatin in plasma and tumor by high-performance liquid chromatography-mass spectrometry

Carboplatin is a platinum analogue that is used in a number of chemotherapeutic regimens for solid tumors, such as lung and ovarian carcinomas. Most often characterization of carboplatin's pharmacokinetic properties is based on measurement of platinum, rather than intact carboplatin. We have developed a sensitive LC-MS method for the determination of intact carboplatin in plasma ultrafiltrate and in tumor tissue. Carboplatin was extracted from rat plasma ultrafiltrate and tumor samples using solid-phase extraction cartridges and analyzed using reversed-phase chromatography with positive electrospray ionization followed by mass spectrometric detection. Using 50 microliter of plasma ultrafiltrate or 140 microliter of tumor homogenate supernatant, the extraction afforded a recovery of 58.7 and 45.8% for plasma and tumor, respectively. The mobile phase was 5% acetonitrile in 0.5% acetic acid at 0.2 ml/min that yielded a retention time of carboplatin of 2.2 min. The method has been validated at carboplatin plasma ultrafiltrate concentrations from 0.07 to 2.5 microgram/ml, and from 0.03 to 1.3 microgram/ml in tumor homogenates. The main advantages of this method compared with earlier methods are the ability to measure intact carboplatin in a sensitive and specific manner.     

Determination of dihydroxynaphthalenes in human urine by gas chromatography-mass spectrometry

A gas chromatography-mass spectrometry (GC-MS) method was developed for measuring 1,2-dihydroxynaphthalene (1,2-DHN) and 1,4-dihydroxynaphthalene (1,4-DHN) in urine. The method involves enzymatic digestion of urinary conjugates to release the DHNs which were then analyzed as trimethylsilyl derivatives by GC-MS. For 1,2-DHN and 1,4-DHN, respectively, the assay limits of detection were 0.21 and 0.15 microg/l, the assay limits of quantitation were 0.69 and 0.44 microg/l, and the coefficients of variation were 14.7 and 10.9%. This method was successfully applied to determine urinary levels of 1,2-DHN and 1,4-DHN in coke workers (14 top workers and 13 side-bottom workers) and 21 matching control workers from the steel industry of northern China. The geometric mean (GM) levels of 1,2-DHN were approximately 100 and 30 times higher than those of 1,4-DHN in exposed and control subjects, respectively. The GM levels 1,2-DHN and 1,4-DHN were significantly higher for coke workers (1,2-DHN: top workers--552 microg/l, side-bottom workers--260 microg/l; 1,4-DHN: top workers--3.42 microg/l, side-bottom workers--3.56 microg/l) than for controls (1,2-DHN: 38.8 microg/l; 1,4-DHN: 1.21 microg/l) (por=0.623; p<0.0001). Also, levels of 1,2-DHN were significantly correlated with those of serum albumin adducts of l,2-naphthoquinone (rs=0.492, p=0.0004). These results indicate that 1,2- and 1,4-DHN are good biomarkers for assessment of naphthalene exposure in coke workers. Since the DHNs are precursors of the naphthoquinones, which have been implicated as toxic products of naphthalene metabolism, measurements of urinary DHNs may have toxicological significance.     

Direct detection of boldenone sulfate and glucuronide conjugates in horse urine by ion trap liquid chromatography-mass spectrometry

A study of the equine phase II metabolism of the anabolic agent boldenone is reported. Boldenone sulfate, boldenone glucuronide and their C17-epimers were synthesised as reference standards in our lab and a method was developed for their detection in a horse urine matrix. Solid phase extraction was used to purify the analytes, which were then detected by ion trap LC/MS. Negative and positive ionisation mode MS(2) were used for the detection of sulfate and glucuronide conjugates, respectively. Boldenone sulfate and 17-epiboldenone glucuronide were detected as the major and minor phase II metabolites, respectively, in horse urine samples collected following the administration of boldenone undecylenate by intramuscular injection.     

Screening for topoisomerase I binding compounds by high-performance liquid chromatography-mass spectrometry

A new rapid compound screening approach for topoisomerase I binding activity is presented. DNA topoisomerase I is used as a target protein to capture binding compounds from a mixture of combinatorial compounds by bioaffinity ultrafiltration. Using high-performance liquid chromatography combined with electrospray ionization mass spectrometry, small-molecule active compounds were identified. We also have successfully applied this method to identifying compounds from cells grown in culture.     

Determination of phenylbutazone and oxyphenbutazone in plasma and urine samples of horses by high-performance liquid chromatography and gas chromatography-mass spectrometry

A method is described for the qualiitative and quantitative determination of phenylbutazone and oxyphenbutazone in horse urine and plasma samples viewing antidoping control. A horse was administered intravenously with 3 g of phenylbutazone. For the qualitative determination, a screening by HPLC was performed after acidic extraction of the urine samples and the confirmation process was realized by GC-MS. Using the proposed method it was possible to detect phenylbutazone and oxyphenbutazone in urine for up to 48 and 120 h, respectively. For the quantitation of these drugs the plasma was deproteinized with acetonitrile and 20 gml were injected directly into the HPLC system equipped with a UV detector and LiChrospher RP-18 column. The mobile phase used was 0.01 M acetic acid in methanol (45:55, v/v). The limit of detection was 0.5 μg/ml for phenylbutazone and oxyphenbutazone and the limit of quantitation was 1.0 μg/ml for both drugs. Using the proposed method it was possible to quantify phenylbutazone up to 30 h and oxyphenbutazone up to 39 h after administration.     

Simultaneous determination of major B-trichothecenes and the de-epoxy-metabolite of deoxynivalenol in pig urine and maize using high-performance liquid chromatography-mass spectrometry

A selective analytical method based on high-performance liquid chromatography (HPLC), combined with atmospheric pressure chemical ionisation (APCI-) mass spectrometry (MS), has been developed for simultaneous determination of B-trichothecenes and the major metabolites of deoxynivalenol. The method allows simultaneous analysis of nivalenol (NIV), deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-AcDON), 3-acetyldeoxynivalenol (3-AcDON), fusarenon X (Fus-X) and de-epoxydeoxynivalenol (DOM-1). The method is based on one-step sample clean-up using a multifunctional MycoSep column. A linear gradient mobile phase system, consisting of water:acetonitrile:methanol (H2O:ACN:MeOH) at a flow-rate of 1 ml/min, and a Polar-RP C18 column, were utilised to obtain the best resolution of all tested compounds along with column and equilibrating within 30 min. Dexamethasone (Dex) was used as internal standard. The developed method shows good repeatability for inter- and intra-day precisions as well as good linearity of calibration curves (r2 ranged from 0.9936 to 0.9998). Average recoveries for tested compounds in both matrices have been determined ranging from 63.7 to 102.3% and limit of quantification (LOQ) ranged from 25 to 150 ng/g. The utility and practical impact of the method is demonstrated using contaminated pig urine and maize samples.     

Development and validation of a liquid chromatography-mass spectrometry assay for the determination of pyronaridine in human urine

A reliable method has been developed for the determination of pyronaridine in human urine using amodiaquine as an internal standard. Liquid-liquid extraction was used for sample preparation. Analysis was performed on a Shimadzu LCMS-2010 in single ion monitoring positive mode using atmospheric pressure chemical ionization (APCI) as an interface. The extracted ion for pyronaridine was m/z 518.20 and for amodiaquine was m/z 356.10. Chromatography was carried out using a Gemini 5 microm C18 3.0 mmx150 mm column using 2 mM perflurooctanoic acid and acetonitrile mixture as a mobile phase delivered at a flow rate of 0.5 mL/min. The mobile phase was delivered in gradient mode. The retention times of pyronaridine and amodiaquine were 9.1 and 8.1 min respectively, with a total run time of 14 min. The assay was linear over a range of 14.3-1425 ng/mL for pyronaridine (R2>or=0.992, weighted 1/Concentration). The analysis of quality control samples for pyronaridine at 28.5, 285, 684 and 1140 ng/mL demonstrated excellent precision with relative standard deviation of 5.1, 2.3, 3.9 and 9.2%, respectively (n=5). Recoveries at concentrations of 28.5, 285, 684 and 1140 ng/mL were all greater than 85%.This LC-MS method for the determination of pyronaridine in human urine has excellent specifications for sensitivity, reproducibility and accuracy and can reliably quantitate concentrations of pyronaridine in urine as low as 14.3 ng/mL. The method will be used to quantify pyronaridine in human urine for pharmacokinetic and drug safety studies.     

Determination of catechins in human urine subsequent to tea ingestion by high-performance liquid chromatography with electrochemical detection

The title determination was conducted by HPLC with electrochemical detection using an ODS column and a mobile phase of acetonitrile: 0.1 M phosphate buffer (pH 2.5) (15:85, v/v). The eight catechins, gallocatechin (GC), epigallocatechin (EGC), catechin (C), epicatechin (EC), epigallocatechin gallate (EGCg), gallocatechin gallate (GCg), epicatechin gallate (ECg), and catechin gallate (Cg), were detected at 0.6 V vs Ag/AgCl. Good linear relationships between current and amount were noted for 0.5-250 pmol of each catechin, with a correlation coefficient of 0.999 in each case. The detection limit for any one was 0.5 pmol (signal to noise ratio, S/N = 3). After the ingestion of 340 ml canned green tea, GC, EGC, C, and EC, mostly in conjugated form, were determined in urine samples. Conjugated catechins were hydrolyzed by enzymes using sulfatase and beta-glucuronidase. The time courses of the above four catechins showed a maxima at 1-3 h after tea ingestion. (+), (-)-EC and (+), (-)-C were present in canned tea.     

Determination of dihydroergocryptine in human plasma and urine samples using on-line sample extraction-column-switching reversed-phase liquid chromatography-mass spectrometry

A rapid and sensitive assay for the determination of dihydroergocryptine (DHEC) in human plasma and urine samples with dihydroergotamine (DHET) as the internal standard was developed. The procedure employs on-line sample preparation using an extraction pre-column and an octadecylsilylsilica (ODS) analytical column. After centrifugation human plasma or urine were injected onto the pre-column, concentrated and extracted, back-flushed onto the analytical column and eluted with a binary methanol--aqueous formic acid gradient. Either determination of DHEC as well of its mono- and dihydroxy-metabolites was performed by measurement of the signal responses from MS detection in the selected reaction monitoring (SRM) mode using the transition of the respective parent ions to the common daughter ion at m/z=270.2 amu. The limit of quantitation (LOQ) for determinations of DHEC in both plasma and urine were 25 pg/ml for injected sample volumes of 400 microl. Proportionality of signal responses versus concentration was accomplished within the range of 25-1000 pg/ml. Recovery of target analyte from plasma was 99%. Mean values of the coefficients of variation (CV) for the target analyte in plasma ranged from 1.7 to 13.8% (within-day) and 5.0 to 9.1% (between-day) and accuracy from 91.7 to 102.6% for the within-day and from 95.8 to 98.8% for the between-day measurements. The corresponding values for determinations in urine were 1.7-14.5% (within-day) and 5.3-11.8% (between-day) for CV and 95.8-110.7% (within-day) and 100.1-104.6% (between-day) for accuracy.