Ml Of Human Serum Research Articles

Simultaneous quantification of endogenous biomarkers and detection of testosterone esters in human serum using gas chromatography-high-resolution mass spectrometry.

High-resolution mass spectrometry (HRMS) has been demonstrated to be an alternative platform for quantitative analyses, identifying unknown compounds and gathering information for the elucidation of chemical structures. This work describes a method to detect 13 esters of testosterone (T) and 5 biomarkers in 0.1mL of human serum using gas chromatography (GC) coupled to HRMS. Analytes were extracted from serum after deproteinization and liquid-liquid extraction. The trimethylsilyl derivatives were analyzed using a gas chromatograph coupled to HRMS at low electron energy to minimize molecule fragmentation. The acquisition in profiling full-scan mode was applied with a resolving power of 30 000 at m/z 400. Linearity, lower limit of quantitation, and measurement uncertainty were assessed. Precision and accuracy were assessed at 0.5 and 2ng/mL, respectively. Mass accuracy (MA) and mass extraction window (MEW) were also evaluated. T esters showed a linear response between 0.25 and 10ng/mL (except for undecanoate, enanthate, and propionate that showed lineal responses between 0.5 and 10ng/mL and isocaproate between 2 and 10ng/mL); detection limits remained between 0.1 and 0.5ng/mL and accuracy between 81% and 119%. The MA (MEW = 10ppm) was maintained between -2.4 and 4.8ppm. The biomarkers (T, androstenedione, dehydroepiandrosterone [DHEA], estradiol, and 17-OH-progesterone) showed a linear response within the evaluated range; quantification limits remained between 0.1 and 0.5ng/mL (except for DHEA), the accuracy between 88% and 99%, and precision between 3.5% and 10.8%. Measurement uncertainties were found between 5.6% and 17.2%. MA (MEW = 3ppm) was maintained between -0.47 and 0.12 ppm. The method to detect T esters and five endogenous biomarkers in serum using GC coupled to HRMS showed linear responses up to 10ng/mL with adequate precision, accuracy, and uncertainties. It was possible to distinguish cholesterol from T-isocaproate based on the MEW of 10ppm, preventing false positives. In addition, this method allows searching for other biomarkers and/or unknown metabolites and other ester forms not included here but at a later stage if necessary.

Electrochemical Genosensing of Overexpressed GAPDH Transcripts in Breast Cancer Exosomes.

Exosomes are receiving highlighted attention as new biomarkers for the detection of cancer since they are profusely released by tumor cells in different biological fluids. In this paper, the exosomes are preconcentrated from the serum by immunomagnetic separation (IMS) based on a CD326 receptor as a specific epithelial cancer-related biomarker and detected by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts. Following the lysis of the captured exosomes, the released GAPDH transcripts are amplified by reverse transcription polymerase chain reaction (RT-PCR) with a double-tagging set of primers on poly(dT)-modified-MPs to increase the sensitivity. The double-tagged amplicon is then quantified by electrochemical genosensing. The IMS/double-tagging RT-PCR/electrochemical genosensing approach is first demonstrated for the sensitive detection of exosomes derived from MCF7 breast cancer cells and compared with CTCs in terms of the analytical performance, showing an LOD of 4 × 102 exosomes μL-1. The genosensor was applied to human samples by immunocapturing the exosomes directly from serum from breast cancer patients and showed a higher electrochemical signal (3.3-fold, p < 0.05), when compared with healthy controls, suggesting an overexpression of GAPDH on serum-derived exosomes from breast cancer patients. The detection of GAPDH transcripts is performed from only 1.0 mL of human serum using specific magnetic particles, improving the analytical simplification and avoiding ultracentrifugation steps, demonstrating to be a promising strategy for minimal invasive liquid biopsy.

Development and validation of a sensitive LC-MS/MS method for the estimation of scopolamine in human serum

Scopolamine is an anticholinergic alkaloid that is widely used in the form of a transdermal system to manage nausea associated with motion sickness. Currently available methods to quantify scopolamine require large sample volumes and involve cumbersome sample preparation. In this work, a simple method for the rapid separation and sensitive quantification of scopolamine in human serum was developed. Scopolamine was extracted from 0.5 mL of human serum using solid-phase extraction. The extracted samples were injected onto Zorbax XDB-C18 column (4.6 × 50 mm, 1.8 μm, and 600 bar) on an Agilent 1200 series HPLC. The chromatographic separation involved gradient elution with water and acetonitrile containing 0.1% v/v formic acid as a mobile phase. The samples were quantified in positive ion mode using a TSQ Quantum triple quadrupole mass spectrometer. The assay was validated and found to be linear over a concentration range of 5–5000 pg/mL. The total assay precision and accuracy was 6.3% and 96%, respectively. The lower limit of quantification (LLOQ) of the assay was 5 pg/mL. The assay was used in a human pharmacokinetic study to measure the concentration of scopolamine in serum after an administering scopolamine as transdermal delivery system or as an intravenous bolus dose.

High-Performance Chemical Isotope Labeling Liquid Chromatography Mass Spectrometry for Exosome Metabolomics.

Circulating exosomes in bodily fluids such as blood are being actively studied as a rich source of chemical biomarkers for cancer diagnosis and monitoring. Although nucleic acid analysis is a primary tool for the discovery of circulating biomarkers in exosomes, metabolomics holds the potential of expanding the chemical diversity of biomarkers that may be easy and rapid to detect. However, only trace amounts of exosomes can be isolated from a small volume of patient blood, and thus a very sensitive technique is required to analyze the metabolome of exosomes. In this report, we present a workflow that involves multiple cycles of ultracentrifugation for exosome isolation using a starting material of 2 mL of human serum, freeze-thaw-cycles in 50% methanol/water for exosome lysis and metabolite extraction, differential chemical isotope labeling (CIL) of metabolites for enhancing liquid chromatography (LC) separation and improving mass spectrometry (MS) detection, and nanoflow LC-MS (nLC-MS) with captivespray for analysis. As a proof-of-principle, we used dansylation labeling to analyze the amine- and phenol-submetabolomes in two sets of exosome samples isolated from the blood samples of five pancreatic cancer patients before and after chemotherapy treatment. The average number of peak pairs or metabolites detected was 1964 ± 60 per sample for a total of 2446 peak pairs ( n = 10) in the first set and 1948 ± 117 per sample for a total of 2511 peak pairs ( n = 10) in the second set. There were 101 and 94 metabolites positively identified in the first and second set, respectively, and 1580 and 1590 peak pairs with accurate masses matching those of metabolites in the MyCompoundID metabolome database. Analyzing the mixtures of 12C-labeled individual exosome samples spiked with a 13C-labeled pooled sample which served as an internal standard allowed relative quantification of metabolomic changes of exosomes of blood samples collected before and after treatment.

Quantification of Folic Acid in Human Serum Using Isotope Dilution Ultra‐High‐Pressure Liquid Chromatography/Mass Spectrometry

Analytical method for an accurate determination of folic acid in human serum has been established using isotope dilution ultra‐high‐pressure liquid chromatography/mass spectrometry (ID‐UPLC/MS). Sample extract was prepared from 0.5 mL of human serum spiked with 13C5‐folic acid solution and then LC–MS/MS experiment was performed on multiple reaction monitoring mode for 20 min. The method was tested for repeatability using homogeneous human serum and it showed good repeatability with relative standard deviation of 1.8%. Further evaluation was carried out through NIST SRM 1955 samples (level I, II, and III) and the measurement results were in good agreement with the certified values with good reproducibility over two periods. It was also revealed that the obtained relative expanded uncertainty was 12% which is much smaller than that of previously reported methods, indicating that this method is very reliable and accurate for determining the content of folic acid in human serum.

Ultrasensitive quantification of serum estrogens in postmenopausal women and older men by liquid chromatography–tandem mass spectrometry

An ultrasensitive stable isotope dilution liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for multiplexed quantitative analysis of six unconjugated and conjugated estrogens in human serum. The quantification utilized a new derivatization procedure, which formed analytes as pre-ionized N-methyl pyridinium-3-sulfonyl (NMPS) derivatives. This method required only 0.1mL of human serum, yet was capable of simultaneously quantifying six estrogens within 20min. The lower limit of quantitation (LLOQ) for estradiol (E2), 16α-hydroxy (OH)-E2, 4-methoxy (MeO)-E2 and 2-MeO-E2 was 1fg on column, and was 10fg on column for 4-OH-E2 and 2-OH-E2. All analytes demonstrated a linear response from 0.5 to 200pg/mL (5–2000pg/mL for 4-OH-E2 and 2-OH-E2). Using this validated method, the estrogen levels in human serum samples from 20 female patients and 20 male patients were analyzed and compared. The levels found for unconjugated serum E2 from postmenopausal women (mean 2.7pg/mL) were very similar to those obtained by highly sensitive gas chromatography–mass spectrometry (GC–MS) methodology. However, the level obtained in serum from older men (mean 9.5pg/mL) was lower than has been reported previously by both GC–MS and LC–MS procedures. The total (unconjugated+conjugated) 4-MeO-E2 levels were significantly higher in female samples compared with males (p<0.05). The enhanced sensitivity offered by the present method will allow for a more specific analysis of estrogens and their metabolites. Our observations might suggest that the level of total 4-MeO-E2 could be a potential biomarker for breast cancer cases.

Simultaneous determination of polybrominated diphenyl ethers and polychlorinated biphenyls by gas chromatography–tandem mass spectrometry in human serum and plasma

Sensitive and reliable methods for simultaneous determination of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in limited volumes of human serum or plasma need to be further documented especially when they accumulate at low levels that are still capable of disrupting endocrine and immune functions, and affecting neurodevelopment and reproduction. The objective of this study was to develop and validate a sensitive and quantitative method that simultaneously quantifies PBDEs and PCBs in 0.5ml of human serum or plasma. We optimized a solid-phase extraction (SPE) method and used silica particle purification for the extraction of PBDEs and PCBs. Two multiple reactions monitoring (MRM) transitions were optimized for each congener. The sum of the transitions was used for quantification, and their abundance ratios were used for identification. The combined method optimization techniques resulted in limits of detection from 3 to 145pg/ml for 10 PBDEs and 1–12pg/ml for 15 PCBs. Method was solidly validated by analyzing serum fortified with a certified PBDE and PCB standard mixture from the National Institute of Standards and Technology (NIST). The accuracy was 88–118% and day-to-day precision was within 19%. The method was successfully applied to quantify native concentrations of PBDE and PCB in commercially available human serum. The sensitivity and selectivity of the GC/EI-MS/MS analysis enables it to be the method of choice for investigations of exposures to PBDE and PCB congeners, especially when sample volume is limited.

Abstract 288: An Improved Method for the Isolation of HDL from Human Serum

In an effort to reduce the time and labor resources required for the standard HDL isolation protocol using sequential ultracentrifugation, our laboratory has investigated a modified process for isolation of HDL from human serum. Our method employs an alternative step for removing VLDL and LDL from human serum that requires less than one hour and replaces the first 18 hour ultracentrifugation step. This alternative process is based on the well documented ability of polyethylene glycol (PEG) to selectively and irreversibly precipitate apolipoprotein B-containing lipoproteins. Subsequently, both methods then use a 21 hour ultracentrifugation spin to separate HDL from all other non-lipoprotein components of human serum. For this study comparing the methods, 1.5 ml of human serum was used. The human serum starting material had a total cholesterol value of 262 mg/dL with the following lipoprotein cholesterol profile: 178 mg/dL LDL-C (68%), 34 mg/dL VLDL-C (13%) and HDL-C 48 mg/dL (18%). Comparison of the two methods demonstrates similar recovery of HDL-C from human serum, 35% vs 37% for the standard method versus modified method, respectively. The total cholesterol recovered using the standard protocol was slightly higher than the modified method. However, as shown by lipoprotein gel electrophoresis, HDL isolated by the standard method was only 80% pure, with 20% of the cholesterol present as VLDL and LDL contaminants remaining in the preparation. In contrast, using our modified process, isolated HDL was &gt;99% pure with no detectable VLDL or LDL cholesterol present. In summary, our laboratory has developed a methodology for the isolation of HDL from human serum that: 1) requires substantially less time and resources, 2) provides the same recovery of HDL-C from serum as the standard method and 3) provides a higher purity HDL than is achieved from the standard sequential ultracentrifugation method.

Quantitative liquid chromatographic and tandem mass spectrometric determination of vitamin D 3 in human serum with derivatization: A comparison of in-tube LLE, 96-well plate LLE and in-tip SPME

Sensitive and selective methods based on high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection were developed for the determination of vitamin D 3 in human serum. Derivatization of vitamin D 3 and its stable isotope labeled internal standard provided highly sensitive quantification and selective detection from endogenous compounds. Samples were prepared using the in-tube liquid–liquid extraction (LLE), 96-well plate LLE, and in-tip solid phase micro-extraction (SPME) in 96-well format. In all methods, the MS/MS detection was performed using Applied Biosystems-Sciex API 3000 tandem mass spectrometers interfaced with a heated nebulizer probe and operated in the positive ionization mode. Both tube and plate LLE methods achieved a lower limit of quantitation (LLOQ) of 0.5 ng/mL when 1.0 and 0.4 mL of human serum was processed, respectively, and were validated in the concentration range of 0.5–25 ng/mL; while for the in-tip SPME method, LLOQ was 5 ng/mL with only 0.1 mL of human serum required. Comparisons were made among three different methods, including precision and accuracy, sample throughput, recovery and matrix effects.

PHARMACOKINETIC STUDY OF RISPERIDONE: APPLICATION OF A HPLC METHOD WITH SOLID PHASE EXTRACTION

A new, simplified solid phase extraction procedure for the determination of risperidone and 9-hydroxyrisperidone in human plasma has been developed. This method involves the use of an optimized extraction protocol developed in Waters OASIS® HLB 30mg 1cc extraction columns using 1 mL of human serum. Separation was performed by HPLC using a Waters XTerra RP-18 (5 µm, 150x4,6 mm) column with a mobile phase consisting in acetonitrile – potassium dihydrogen phosphate 50 mM pH 3.4 (27/73). UV detection at 278 nm was used to quantify analytes, encountering good linearity (r2 > 0.999) in the 2-100 ng/mL concentration range. The mean recovery was 92.4 % and 92.8 % for risperidone and 9-hydroxyrisperidone respectively, with an intraday – interday precision below 7%, and accuracy below 115 %. The method has been successfully applied in pharmacokinetic studies that require a large sample number.

Structure of a neutral glycosphingolipid recognized by human antibodies in polyagglutinable erythrocytes from the rare NOR phenotype.

NOR is a rare inheritable polyagglutination phenomenon that has been described in two families. Our recent studies on these erythrocytes showed they contained at least two unique neutral glycosphingolipids, and based on their reactivity with Griffonia simplicifolia IB4 (GSL-IB4) isolectin (Kusnierz-Alejska, G., Duk, M., Storry, J. R., Reid, M. E., Wiecek, B., Seyfried, H., and Lisowska, E. (1999) Transfusion 39, 32-38), both oligosaccharide chains terminated with an alpha-galactose residue. The reactivity with GSL-IB4 suggested that these oligosaccharide chains terminated with a Galalpha1-->3Gal- sequence and that anti-NOR agglutinins were common human anti-Galalpha1-->3Gal xenoantibodies. In this report we describe the structure of one NOR component (NOR1) that migrated on thin-layer chromatographic plates in the region of pentaglycosylceramides. Treatment of this sample with alpha-galactosidase and beta-N-acetylhexosaminidase was followed by high-performance thin-layer chromatography with product detection by lectins and the anti-Gb4 monoclonal antibody. The results suggested that NOR1 was an alpha-galactosylated Gb4Cer with a beta-N-acetylhexosaminidase-resistant GalNAc residue. Gas phase disassembly by ion trap mass spectrometry analysis showed the sequence to be Hex1-->4HexN1-->3Hex1-->4Hex1-->4Hex linked to a ceramide composed of C18 sphingosine and a C24 monounsaturated fatty acid. Together these data indicate NOR1 to be a novel Galalpha1-->4GalNAcbeta1-->3Galalpha1-->4Galbeta1-->4 Glc-Cer structure. Additionally it has been shown that NOR glycolipids are recognized by human antibodies that were distinct from the known anti-Galalpha1-->3Gal xenoantibodies.

Simultaneous determination of zidovudine and lamivudine in human serum using HPLC with tandem mass spectrometry

A method employing high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS) has been developed and validated for the simultaneous determination of clinically relevant levels of zidovudine (AZT) and lamivudine (3TC) in human serum. The method incorporates a fully automated ultrafiltration sample preparation step that replaces the solid-phase extraction step typically used for HPLC with UV detection. The calibration range of the dual-analyte LC-MS/MS method is 2.5–2500 and 2.5–5000 ng ml −1 for AZT and 3TC, respectively, using 0.25 ml of human serum. The lower limit of quantification was 2.5 ng ml −1 for each analyte, with a chromatographic run time of approximately 6 min. Overall accuracy, expressed as bias, and inter- and intra-assay precision are <±7 and <10% for AZT, and <±5 and <12.1% for 3TC over the full concentration ranges. A cross-validation study demonstrated that the LC-MS/MS method afforded equivalent results to established methods consisting of a radioimmuno-assay for AZT and an HPLC-UV method for 3TC. Moreover, the LC-MS/MS was more sensitive, allowed markedly higher-throughput, and required smaller sample volumes (for 3TC only). The validated method has been used to support post-marketing clinical studies for Combivir™ — a combination tablet containing AZT and 3TC.

Liquid chromatography/electrospray tandem mass spectrometry method for the quantitation of fosinoprilat in human serum using automated 96-well solid-phase extraction for sample preparation.

A sensitive, specific, accurate and reproducible liquid chromatography/electrospray tandem mass spectrometry method was developed and validated for the quantitation of fosinoprilat in 0.2 mL of human serum. The method employed acidification (with pH 4.0 sodium acetate buffer) of the serum samples to minimize the hydrolysis of the prodrug fosinopril to fosinoprilat prior to purification by automated 96-well solid-phase extraction. The required chromatographic separation of fosinoprilat and fosinopril was achieved isocratically on a Luna C8 analytical column (2 x 50 mm, 3 microm). The total run time was 2 min. The mobile phase contained methanol and water with 10 mM ammonium acetate. Detection was by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 2.00 to 500 ng/mL, was fitted to a 1/x(2) weighted linear regression model. Fosinoprilat quality control (QC) samples used to determine the accuracy and precision of the method were prepared in human serum at concentrations of 5.00, 200, 400 and 1000 ng/mL. The assay accuracy was within 8% (dev). The intra- and inter-assay precisions were within 6 and 3% (RSD), respectively. Fosinopril QC samples used to gauge the rate of hydrolysis of fosinopril to fosinoprilat during the assay procedure were prepared in human serum at 500 ng/mL. The hydrolysis of fosinopril to fosinoprilat was </=1%. This degree of conversion would cause little error in the analysis of post-dose serum samples since such samples are known to contain low levels of the prodrug compared with the drug.

Double column-switching high-performance liquid chromatographic method for the determination of TAK-603 and its metabolites in human serum

A double column-switching high-performance liquid chromatographic (HPLC) method for the determination of concentrations for TAK-603 (T) and its metabolites, T-72258 (M-I) and T-72294 (M-III), in human serum was developed. The analytes were extracted with ethyl acetate from human serum samples treated with triethylamine and injected into the HPLC system. Separation of the analytes was performed on the HPLC system with double column-switching technique. The mobile phases A and B for the first column and the mobile phase C for the second column used were a mixture of methanol–10 m M aqueous ammonium acetate solution (1:1, v/v), methanol and a mixture of methanol–10 m M aqueous ammonium acetate solution (11:9, v/v), respectively. The eluate was monitored with a UV detector at a wavelength of 253 nm. The work-up procedure was reproducible and more than 90% of the analytes could be recovered from human serum. The lower limits of quantitation were all 1 ng/ml for the analytes when 0.5 ml of human serum was used. Standard curves were linear with a correlation coefficient ( R) of more than 0.999 in the range of 1–500 ng/ml for T, M-I and M-III in human serum. The intra- and inter-day precision of the method for the various analytes were below 4.8%. The accuracy was good with the deviations between spiked and calculated concentrations of the analytes being within 11.0%. The method was successfully applied to analyze serum samples after an oral administration of T to healthy male volunteers.

Semi-automatic liquid chromatographic analysis of pamidronate in serum and citrate plasma after derivatization with 1-naphthylisothiocyanate

The semi-automatic method for the determination of the bisphosphonate pamidronate in serum and citrate plasma involves a manual protein precipitation with trichloroacetic acid and a manual coprecipitation of the bisphosphonate with calcium phosphate, followed by an automated solid-phase extraction on anion-exchange columns. After off-line evaporation of the extract under nitrogen and reconstitution in water, the automatic procedure is continued by automatic derivatization with 1-naphthylisothiocyanate, ion-pair liquid–liquid extraction and a treatment with hydrogen peroxide, prior to analysis by ion-pair HPLC and fluorescence detection at 285/390 nm. The intra- and inter-day precisions are 1.3 and 7%, respectively, for a standard of 100 ng ml −1 pamidronate in serum; the average accuracy for this standard is 107%. The lower limit of quantification is 20 ng ml −1 pamidronate in 1 ml of human serum.

Investigative methods of congenital complete heart block

Congenital heart block (CHB) has been described for a century and related to the presence of maternal autoimmune disease for three decades, but little is understood about its mechanism. To explore the pathophysiology of CHB, technologies in both basic and clinical electrophysiology are being developed and applied. Human fetal rhythm is currently inferred from cardiac mechanical events by using fetal ultrasound, allowing for the detection of second and third-degree heart block. Fetal electrocardiography is being explored to assess its feasibility as a clinical tool to detect fetal first-degree block in the mid trimester. Sequential composite digital recordings from the maternal abdomen are made every 4 weeks from pregnancies at risk for congenital heart block. Filtering and averaging techniques are used to enhance the fetal signal. So far, these techniques have produced a fetal QRS complex trigger signal for use in three-dimensional fetal echocardiography. Because the human fetus cannot be studied directly, a Langendorff rabbit model of CHB has been developed. With 5–10 mL of human serum in 150–300 mL of Krebs solution, prolongation of the Wenckebach second-degree atrioventricular block cycle length occurred. This was reproduced by using serum from seven of eight CHB mothers as compared with none of six controls mothers.

Stereoselective determination of R-(-)- and S-(+)-prilocaine in human serum by capillary electrophoresis using a derivatized cyclodextrin and ultraviolet detection.

A capillary electrophoresis (CE) method for the quantification of R-(-)- and S-(+)-prilocaine in human serum was developed and validated. Stereoselective resolution was accomplished using 15 mM heptakis(2,6-di-methyl)-beta-cyclodextrin and 0.03 mM hexadecyltrimethylammonium bromide (HTAB) contained in 100 mM phosphate buffer, pH 2.5. Solid-phase extraction was used as a sample preparation technique to remove endogenous interferences. A 72-cm uncoated fused-silica capillary at a voltage of 25 kV and 30 degrees C was used for the analysis. The detection limits for R-(-)- and S-(+)-prilocaine were 38 ng/ml using 1 ml of human serum and the limits of quantitation were 45 ng/ml. The calibration curve was linear over the range of 45-750 ng/ml with procainamide as the internal standard. Precision and accuracy of the method were 2.86-8.50% and 3.29-7.40%, respectively, for R-(-)-prilocaine, and 3.94-9.17% and 2.0-6.73%, respectively, for S-(+)-prilocaine. The CE method was compared to an existing chiral HPLC method in terms of sensitivity and selectivity for the routine analysis of the drug.

Pyrene sulphonyl chloride as a reagent for quantitation of oestrogens in human serum using HPLC with conventional and laser-induced fluorescence detection.

A highly sensitive HPLC method with conventional and laser-induced fluorescence detection for the analyses of oestrogens is described. beta-oestradiol (beta-ES) was chosen as a model and derivatized with pyrenesulphonyl chloride (PSCL), a novel derivatizing reagent, using a two-phase system with tetrapentylammonium bromide (TPABR) as a phase transfer catalyst. Derivatization was complete in 30 s at room temperature and concentrations as low as 5 x 10(-10) M could be derivatized and detected. The concentration detection limit PSCL derivatized beta-ES was 2 x 10(-11) M which corresponds to an on-column detection limit of one femtomole using conventional chromatography and detection. The relative standard deviation (n = 6) of the derivatization carried out at 2.5 x 10(-9) M was 4%. No significant loss of peak-height of the derivative was observed at room temperature over 24 h period. Baseline resolution of PSCL derivatized oestrone, equilin and beta-oestradiol was achieved with ACN:H2O (75:25 v/v) using a Bondclone C-18 column. The method described here is sufficiently sensitive for analyses of beta-oestradiol at low pg/mL concentrations in 1 mL of human serum. The PSCL derivatives of oestrogens demonstrate excellent potential for excitation by laser-induced fluorescence using the 351 nm line of an Ar ion laser or the 325 nm line of He-Cd laser. A concentration limit of detection of 4 x 10(-12) M for beta-oestradiol was achieved using the 325 nm line of an He-Cd laser. This corresponds to 0.2 fmol of derivatized beta-oestradiol on-column.

Quantitative determination of amlodipine in serum by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry.

A sensitive and specific liquid chromatographic method coupled with tandem mass spectrometry was developed for the quantification of amlodipine in human and rat serum, which is a dihydropyridine derivative with calcium antagonist activity. An atmospheric pressure chemical ionization interface was used as the ion source and the analysis was performed in the selected reactive monitoring (SRM) mode. Deuterated amlodipine was used as the internal standard, and serum samples were treated with diethyl ether extraction prior to analysis. Serum levels in the range 0.014-7.2 ng ml-1 were measured accurately by this method, and the lower limit of quantitation (LOQ) was 0.014 ng ml-1 using 1 ml of human serum. The accuracy was within 7% of the expected values. The intra-assay precision was less than 3% and the inter-assay precision was less than 6%. The method was applied to a pharmacokinetic study of amlodipine in rats, in which the measurable range was 0.14-72 ng ml-1 using 0.1 ml of serum because of a limitation on the sample volume.

Simultaneous determination of deflazacort metabolites II and III, cortisol, cortisone, prednisolone and prednisone in human serum by reversed-phase high-performance liquid chromatography.

A new high-performance liquid chromatographic method for the analysis of human serum from subjects administered prednisolone or deflazacort has been established. Two deflazacort metabolites (metabolites II and III), prednisolone and its metabolite (prednisone), as well as cortisol and cortisone in human serum were analyzed simultaneously. These six compounds and the internal standard, betamethasone, were extracted from 1 ml of human serum using a Sep-Pak Plus Environmental C18 column and then separated on a Hypersil ODS (25 x 0.46 cm I.D.) column using a gradient system. The lower limit of quantitation for each compound was 10 ng/ml based on signal-to-noise ratio of 5 and calibration curve linearity. Within-day and between-day validations gave a coefficient of variation of less than 10% and a relative error of ca. 10% (n = 11).