Bio-compatible in-tube solid-phase microextraction capillary for the direct extraction and high-performance liquid chromatographic determination of drugs in human serum

A restricted access material (RAM), alkyl-diol-silica (ADS), was used to prepare a highly bio-compatible solid-phase microextraction (SPME) capillary for the automated and direct in-tube extraction of several benzodiazepines from human serum. The bifunctionality of the ADS extraction phase prevented fouling of the capillary by protein adsorption while simultaneously trapping the analytes in the hydrophobic porous interior. This the first report of a restricted access material utilized as an extraction phase for in-tube SPME. The approach simplified the required apparatus in comparison to existing RAM column switching procedures, and more importantly eliminated the excessive use of extraction solvents. The biocompatibility of the ADS material also overcame the existing problems with in-tube SPME that requires an ultrafiltration or other deproteinization step prior to handling biological samples, therefore further minimizing the sample preparation requirements. The calculated oxazepam, temazepam, nordazepam and diazepam detection limits were 26, 29, 22 and 24 ng/ml in serum, respectively. The method was linear over the range of 50-50 000 ng/ml with an average linear coefficient (R2) value of 0.9998. The injection repeatability and intra-assay precision of the method were evaluated with five injections of a 10-microg/ml serum sample (spiked with all compounds), resulting in an average RSD<7%. The ADS extraction column was robust, providing many direct injections of biological fluids for the extraction and subsequent determination of benzodiazepines.     

Determination of selenium in human serum by liquid chromatography/electron capture atmospheric pressure chemical ionization mass spectrometry after acid digestion and derivatization using 2,3-diaminonaphthalene

Analysis of selenium in biological samples is very important and numerous analytical methods for the element have been developed. One of the most convenient and widely used methods for routine determination of serum selenium is a fluorometric method using 2,3-diaminonaphthalene (DAN); however, this method lacks specificity. We observed that 4,5-benzopiazselenol (BPS), a selenium derivative of DAN, is ionized with electron capture in an atmospheric pressure chemical ionization (APCI) interface, and subsequently established a method for determining total human serum selenium by means of liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. All pretreatment procedures were carried out in a single test tube to minimize selenium loss. The recovery of organic or inorganic selenium spiked to human serum was 97-103%.The detection limit of BPS was equivalent to 0.2 ng of selenium and the lower quantitative limit of serum selenium was 10 ng mL(-1). The coefficient of variation of standard concentrations in control serum samples was 4.5%. The purity of the observed peak obtained from serum samples was confirmed using the ion cluster technique.     

Sensitive method for the determination of the antiarrhythmic drug detajmium in serum by solid-phase extraction and high-performance liquid chromatography with fluorimetric detection.

The objective of this study was to develop a very sensitive and selective method for the determination of detajmium (4-3-diethylamino-2-hydroxypropyl-ajmaline), a sodium-channel-blocking drug with antiarrhythmic properties, in serum. A high-performance liquid chromatography (HPLC) method with solid-phase extraction and fluorimetric detection has been applied. Serum samples were diluted with phosphate buffer (pH 3.5) and the extraction of detajmium and ajmaline, which was used as an internal standard, was carried out with Oasis cartridges (Waters). The chromatographic separation was performed on a RP18 column. The limit of quantification for serum samples of detajmium was 1 ng/ml with good reproducibility (R.S.D. < 15%) and a linear response from 1 to 200 ng/ml. The described method is highly sensitive and specific for the determination of detajmium in serum of patients and volunteers.     

磁性反相限进材料的制备及其应用

以磁性二氧化硅（Fe₂O₃@SiO₂）为基质,利用表面原子转移自由基聚合技术（SI-ATRP）,在改性后的Fe₂O₃@SiO₂内表面接枝甲基丙烯酸十八烷基酯（SMA）、外表面接枝甲基丙烯酸缩甘油酯（GMA）,酸解后得到磁性反相限进材料Fe₂O₃@SiO₂-SMA-GMMA,并通过扫描电镜（SEM）、X射线衍射（XRD）、透射电镜（TEM）、振动样品磁强计（VSM）和元素分析对其进行表征。研究表明,Fe₂O₃@SiO₂-SMA-GMMA对牛血清白蛋白（BSA）的排阻能力为90.4%;对磺胺异恶唑（SIZ）、磺胺二甲氧基嘧啶（SDM）、甲氧苄啶（TMP）和磺胺甲基嘧啶（SMR）的最大吸附量分别为2.76、2.24、1.51和1.34 mg/g。Fe₂O₃@SiO₂-SMA-GMMA应用于牛奶和牛血清样品中SIZ、SMR和SDM的分离富集,SIZ、SMR和SDM的加标回收率为88.7%~90.8%,相对标准偏差为3.3%~5.3%。磁性反相限进材料可简化生物基质样品的前处理过程,对血液样品或食品样品等领域的分析检测具有重要价值。     

Simultaneous quantification of sulfamethoxazole and trimethoprim in whole egg samples by column-switching high-performance liquid chromatography using restricted access media column for on-line sample clean-up

This work reports the application of restricted access media (RAM) column, in a multidimensional configuration, for simultaneous analysis of sulfamethoxazole (SMX) and trimethoprim (TMP) in whole eggs with ultraviolet detection. The proteins were partially precipitated by adding 0.5 mL of acetonitrile into 1.0 mL of blended egg followed by centrifugation. The supernatant was injected (250 microL) directly into the multidimensional system. At the first dimension, a restricted access medium (RAM) bovine serum albumin (BSA) octadecyl column (100 mm x 46 mm I.D., Luna silica, 10 microm particle size and 100 A pore size), was used for extraction and concentration of the analytes and at second dimension, an octadecyl column (150 mm x 46 mm I.D., Luna silica, 10 microm particle size and 100 A pore size), for analysis. The developed method showed good selectivity, accuracy and precision for quantification of these different compounds in eggs, and the limits of quantification were 80 ng/mL, for both compounds. The validated method is reliable and sensitive for monitoring residues in whole eggs samples and thus, to determine withdraw period for laying hens using veterinary medicine having SMX-TMP combination.     

Continuous-flow enzymatic determination of creatinine with improved on-line removal of endogenous ammonia

A new continuous-flow automated enzymatic method suitable for the direct determination of creatinine in physiological samples is described. The proposed system utilizes an on-line gas predialysis unit in conjuction with a flow-through enzyme reactor coil and a potentiometric ammonia detector. The enzyme reactor contains immobilized creatinine iminohydrolase (EC 3.5.4.21) which converts creatinine to ammonia and N-methylhydantoin. Ammonia liberated from this reaction is detected downstream with the membrane electrode-based detector. The novel gas predialysis unit effectively removes >99.8% of endogenous ammonia (up to 1 mM) present in the sample. Thus, final peak potentials recorded by the electrode detector are directly proportional to the logarithm of creatinine concentrations present. The method is shown to be precise (<3%), selective, and capable of accurately determining creatinine in serum and urine samples containing abnormally high endogenous ammonia levels. Determinations of creatinine in serum samples (n = 30) using this new method correlate well with an existing Technicon AutoAnalyzer colorimetric method (r = 0.996).     

New contributions to the field of bead-injection spectroscopy—flow-injection analysis: determination of cobalt

A novel method based on column-switching high-performance liquid chromatography-electrospray mass spectrometry (LC-MS) coupled with an on-line extraction column containing conjugated avidin has been developed for direct injection analysis of di(2-ethylhexyl) phthalate (DEHP) and its metabolite, mono(2-ethylhexyl) phthalate (MEHP), in blood samples. The sample preparation for on-line extraction involved the mixing of blood samples with internal standards, DEHP-d(4) and MEHP-d(4), in LC glass vials. A linear response was found for column-switching LC-MS when tests were conducted within the validated range of 25 to 1000 ng mL(-1) for DEHP and 5 to 1000 ng mL(-1) for MEHP, with correlation coefficients (r) greater than 0.999. In addition, the recoveries of DEHP and MEHP from human plasma were calculated by using this method with on-line extraction, yielding recoveries of up to 91.2% (RSD<5%). We measured the background levels of DEHP and MEHP in six human plasma samples from healthy volunteers and three fetal bovine serum samples for cell-line culture. DEHP and MEHP were not detected in all human plasma samples (N.D. is <25 ng mL(-1) for DEHP, and N.D. is <5.0 ng mL(-1) for MEHP). In contrast, high DEHP contamination of commercially available fetal bovine serum samples was found by this method.     

Enzymatic determination of cholesterol and triglycerides in serum lipoprotein profiles by asymmetrical flow field-flow fractionation with on-line, dual detection

Alterations of lipoproteins (LPs) and related lipid levels in blood serum are correlated to the risk of coronary artery disease (CAD). Fast, possibly automated methods to obtain complete, multi-parametric LP profiles are therefore welcome to be developed for routine, clinical analysis practice. In this work, asymmetrical flow field-flow fractionation (AF4) with on-line, dual post-fractionation reaction detection (PFRD) is applied to develop a method for single-run, simultaneous quantification of cholesterol (CHOL) and triglycerides (TGs) in each fractionated LP class. The enzymatic reagents used for the post-fractionation reaction are available as commercial kits for certified, standard clinical protocols for the analysis of CHOL and TGs in serum. Using CHOL and glycerol as reference standards, a new procedure is applied to optimize the experimental conditions for PFRD-based, quantitative analysis. Upon optimized PFRD and AF4 conditions, results obtained for the determination of total CHOL (TC), TGs, HDL-cholesterol (HDL-C), and LDL-cholesterol (LDL-C) in a set of serum samples from healthy donors are found in agreement with the values provided by a clinical laboratory. The intra-day and inter-day precisions of the method were found always lower than 10% (CV). When the method was applied to serum samples from patients affected by sepsis, differences in CHOL and TG profiles between patients and healthy donors were observed.     

Optimised Method for Short-Chain Fatty Acid Profiling of Bovine Milk and Serum

Short-chain fatty acids (SCFA, C2-C5) in milk and serum are derived from rumen bacterial fermentation and, thus, have the potential to be used as biomarkers for the health status of dairy cows. Currently, there is no comprehensive and validated method that can be used to analyse all SCFAs in both bovine serum and milk. This paper reports an optimised protocol, combining 3-nitrophenylhydrazine (3-NPH) derivatisation and liquid chromatography-mass spectrometry (LC-MS) analysis for quantification of SCFA and β-hydroxybutyric acid (BHBA) in both bovine milk and bovine serum. This method is sensitive (limit of detection (LOD) ≤ 0.1 µmol/L of bovine milk and serum), accurate (recovery 84–115% for most analytes) and reproducible (relative standard deviation (RSD) for repeated analyses below 7% for most measurements) with a short sample preparation step. The application of this method to samples collected from a small cohort of animals allowed us to reveal a large variation in SCFA concentration between serum and milk and across different animals as well as the strong correlation of some SCFAs between milk and serum samples.     

Ion-Paired Liquid Chromatographic Determination of Phenylglyoxal Bis(Guanylhydrazone) in Serum and Urine

Abstract

Phenylglyoxal bis(guanylhydrazone) (PGBG),shows a strong inhibitory activity of the same order and pattern as Methylglyoxal bis (guanylhydrazone) (MGBG), against Diamine oxidase from the small intestine of the rats and against S-adenosylmethionine decarboxylase (one of the key enzymes of polyamine biosinthesis) from Eimeria stiedai cysts, and from partially purified rabbit liver enzyme. The therapeutic interest of polyamine biosynthesis inhibitors in tumoral, infectious and parasitic processes and the lesser toxicity of PGBG than of MGBG favour the use of this compound as an alternative treatment for this kind of disease.

For this study a paired-ion HPLC method was developed to measure concentrations of PGBG in serum and urine samples. The method involved ultrafiltration of diluted serum and urine through a 30,000-Dalton molecular weight cut-off filter.

Recoveries for PGBG in serum and urine were 97–99%. Ultrafiltered samples were then injected into a C₁₈ column and eluted isocratically with a mobile phase consisting of a mixture of phosphate buffer 0.05 M, pH=3.5, methanol (83:17) and 1.5 mM a simple ultrafiltration sample, Ultrafree-MC, system with a molecular weight cut-off of 30,000 Daltons.

The proposed method is practical quick and accurate and requires small sample volumes to determine PGBG in serum and urine.     

Determination of theophylline in serum and saliva in the presence of caffeine and its metabolites.

Because of marked variability in its metabolic clearance and its narrow therapeutic range (10-20 micrograms/ml) investigation of each patient's clearance of theophylline is desirable. The author reports here a rapid reversed-phase high-performance liquid chromatographic (HPLC) method to determine, within 3 min, the theophylline in serum and saliva in the 0.1-50 micrograms/ml range. A fast HPLC column, 10 x 4.6 mm, packed with 3-microns spherical ODS packing is used with acetonitrile-methanol-buffer pH 4.7 (4:7:89) to achieve separation of theophylline from paraxanthine and matrix components. Since theophylline is a major pediatric bronchodilator, the feasibility of assay in saliva was investigated as an alternative route for determining the clearance is stressed asthmatic children. Using this method it was found that the ratio of theophylline in simultaneous serum and saliva samples is very consistent over time in the same person (+/- 3.99%), but inter-individually this consistency is reduced ten-fold. Simultaneous serum and saliva samples need be taken only once to obtain the ratio and the kinetics followed further with salivary samples only.     

Simultaneous Biparametric Determination of Total Calcium and Potassium in Biological Fluids by Flow Injection Analysis──Use of Powell＇s Method in the System Optimization

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Glycoprofiling as a novel tool in serological assays of systemic sclerosis: A comparative study with three bioanalytical methods

Systemic sclerosis (SSc) is an autoimmune disease seriously affecting patient’s quality of life. The heterogeneity of the disease also means that identification and subsequent validation of biomarkers of the disease is quite challenging. A fully validated single biomarker for diagnosis, prognosis, disease activity and assessment of response to therapy is not yet available. The main aim of this study was to apply an alternative assay protocol to the immunoassay-based analysis of this disease by employment of sialic acid recognizing lectin Sambucus nigra agglutinin (SNA) to glycoprofile serum samples. To our best knowledge this is the first study describing direct lectin-based glycoprofiling of serum SSc samples. Three different analytical methods for glycoprofiling of serum samples relying on application of lectins are compared here from a bioanalytical point of view including traditional ELISA-like lectin-based method (ELLA), novel fluorescent lectin microarrays and ultrasensitive impedimetric lectin biosensors. Results obtained by all three bioanalytical methods consistently showed differences in the level of sialic acid present on glycoproteins, when serum from healthy people was compared to the one from patients having SSc. Thus, analysis of sialic acid content in human serum could be of a diagnostic value for future detection of SSc, but further work is needed to enhance selectivity of assays for example by glycoprofiling of a fraction of human serum enriched in antibodies for individual diagnostics.     

Spectrofluorimetric determination of anthranilic acid derivatives based on terbium sensitized fluorescence.

Terbium sensitized fluorescence was used to develop a sensitive and simple spectrofluorimetric method for the determination of the anthranilic acid derivatives furosemide and mefenamic and tolfenamic acids. The method makes use of radiative energy transfer from anthranilates to terbium ions in alkaline methanolic solutions. Optimum conditions for the formation of the anthranilate-Tb3+ complexes were investigated. Under optimized conditions, the detection limits are 6 x 10(-9), 1.4 x 10(-8) and 9.0 x 10(-9) mol l-1 for furosemide, mefenamic acids and tolfenamic acid, respectively. The range of application is 2.5 x 10(-8)-5.0 x 10(-5) mol l-1 for all three drugs. The method was successfully applied to the determination of furosemide and mefenamic and tolfenamic acids in serum after extraction of the samples with ethyl acetate, evaporation of the organic layer under a stream of nitrogen at 40 degrees C and reconstitution of the residue with alkaline methanolic terbium solution prior to instrumental measurement. The mean recoveries from serum samples spiked with furosemide (5.0 x 10(-7), 2.0 x 10(-6) and 8.0 x 10(-6) mol l-1), mefenamic acid (3.0 x 10(-6), 9.0 x 10(-6) and 3.0 x 10(-5) mol l-1) and tolfenamic acid (3.1 x 10(-6), 12.5 x 10(-6) and 2.5 x 10(-5) mol l-1) were 96 +/- 8, 101 +/- 5 and 98 +/- 7%, respectively. The within-run precision (RSD) for the method for two serum samples of each drug varied from 2 to 8% and the day-to-day precision for two concentration levels varied from 2 to 13%.     

Hydrazide-functionalized magnetic microspheres for the selective enrichment of digested tryptophan-containing peptides in serum

The extreme complexity of protein samples is becoming a great challenge for proteomic analysis, especially for those having large dynamic range of protein abundance. To solve this problem, and to overcome the limitation of the current proteomic technologies, a new method using hydrazide-functionalized magnetic microspheres was established in this study. With this method, tryptophan (Trp)-containing peptides can be selectively and sensitively enriched from complex and low-volume samples. Furthermore, combined with 1D-LC-MS/MS analysis, the strategy was successfully applied to the proteomic study of mouse serum. The proportion of Trp-containing peptides was increased from 19.4% to 80.2% through enrichment, and the complexity of the sample was reduced more than two times. An additional 113 Trp-containing peptides and 48 novel proteins were detected compared to the conventional method. This enrichment method provides a means for identifying more proteins as potential biomarkers in serum and other complex samples.     

Detection of cerebrospinal fluid leakage by specific measurement of transferrin glycoforms

A simple and rapid detection of cerebrospinal fluid (CSF) leakage would benefit spine surgeons making critical postoperative decisions on patient care. We have assessed novel approaches to selectively determine CSF β2‐transferrin (β2TF), an asialo‐transferrin (aTF) biomarker, without interference from serum sialo‐transferrin (sTF) in test samples. First, we performed mild periodate oxidation to selectively generate aldehyde groups in sTF for capture with magnetic hydrazide microparticles, and selective removal with a magnetic separator. Using this protocol sTF was selectively removed from mixtures of CSF and serum containing CSF aTF (β2TF) and serum sTF, respectively. Second, a two‐step enzymatic method was developed with neuraminidase and galactose oxidase for generating aldehyde groups in sTF present in CSF and serum mixtures for magnetic hydrazide microparticle capture. After selectively removing sTF from mixtures of CSF and serum, ELISA could detect significant TF signal only in CSF, while the TF signal in serum was negligible. The new approach for selective removal of only sTF in test samples will be promising for the required intervention by a spine surgeon.     

Liquid chromatographic method with electrochemical detection for determination of cisapride in serum

A sensitive liquid chromatographic (LC) method using electrochemical detection was developed for the identification and quantitation of cisapride in serum. The serum samples were deproteinized by a simple acetonitrile precipitation technique followed by n-hexane extraction. Cisapride in the deproteinized serum was separated by an isocratic elution with an ODS Hypersil LC column (150 x 4.6 mm) using a mobile phase consisting of 0.05M Na2HPO4-acetonitrile (60 + 40), pH 8.4. Cisapride eluted from the column was detected by a Coulochem II electrochemical detector. The precision of this assay method was determined by intra- and inter-day analyses of cisapride-free fetal bovine serum samples that were spiked with 25, 50, and 100 ng/mL cisapride. For the intra-day assay, recoveries were 94.3 +/- 1.4, 90.1 +/- 2.9, and 103.2 +/- 9.2%, respectively. This electrochemical detection LC method could be very useful in monitoring plasma levels of cisapride.     

Protein–Incorporated Serum Total Antioxidant Capacity Measurement by a Modified CUPRAC (CUPRIC Reducing Antioxidant Capacity) Method

Due to inadequacies in analytical methodology of total antioxidant capacity (TAC) measurement, proteins are initially separated from the human serum matrix by precipitation and are left unmeasured, thereby causing an important “antioxidant gap.” The aim of this work is to measure the TAC of serum with the modified CUPric Reducing Antioxidant Capacity (CUPRAC) method, and to identify the contribution of serum proteins, especially thiol-containing proteins, to TAC. CUPRAC results were statistically compared to those found by reference methods, namely ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), FRAP (ferric reducing antioxidant power), and Ellman thiols assay. The curves of absorbance vs thiol concentration, as well as of absorbance vs diluted serum (whole serum, trichloroacetic acid (TCA)?precipitated and redissolved serum protein solution, and TCA supernatant fractions) volume, of three distinct serum samples showed excellent linearity and low intercept values only with the modified CUPRAC method. The proposed method will help characterize the “antioxidant gap” of serum TAC originating from protein components which should not be neglected in future antioxidant measurements.     

Determination of the ubiquinol-10 and ubiquinone-10 (coenzyme Q10) in human serum by liquid chromatography tandem mass spectrometry to evaluate the oxidative stress

A method for the rapid and simultaneous determination of ubiquinone-10 (coenzyme Q10, CoQ(10)) and the reduced form ubiquinol-10 (CoQ(10)H(2)) in human serum by LC-MS-MS with electrospray ionization (ESI) in the positive mode is here proposed. High selective identification and sensitive quantitation of both analytes have been carried out by monitoring the transition from the corresponding precursor ion to the product ion. Prior to the chromatographic analysis, serum samples (100 microl) were subject to a conventional pre-treatment based on protein precipitation, liquid-liquid extraction, evaporation to dryness and reconstitution with 95:5 methanol/hexane (v/v). The overall method has enabled to achieve low detection limits--5.49 and 15.8 ng/ml for CoQ(10) and CoQ(10)H(2), respectively--which were estimated with serum. The accuracy and potential matrix effects have been studied with spiked serum resulting recoveries between 92.82 and 106.97%. The proposed method has been applied to serum samples from healthy middle-age women, in which the CoQ(10)H(2)/CoQ(10) ratio has been used as marker of oxidative stress.     

Evaluation of serum as a potential matrix for multiresidue determination of fluoroquinolone antibiotics in chicken using liquid chromatography-fluorescence-mass spectrometry(n)

An efficient multiresidue method was successfully applied to the determination of fluoroquinolones (FQs) in chicken serum. In this method, FQs are extracted from matrix with ammoniacal acetonitrile, and the extracts are defatted and then evaporated. After addition of basic phosphate buffer and filtration, the samples are analyzed by liquid chromatography-fluorescence-mass spectrometry(n) (multiple mass spectrometry; MS(n)). This approach allows for simultaneous quantitation (fluorescence) and confirmation (MS(n)) of the FQs. Using this method, 8 FQs were determined in fortified chicken serum at levels of 10, 20, 50, and 100 ng/g. Recoveries ranged from 71-99%, with excellent relative standard deviations (< 10%). Limits of quantitation for the FQs ranged from 0.05-5 ng/g. Confirmation was achieved by comparison of MS2 or MS3 product ion ratios with those of standard FQ samples. These quantitative and confirmatory results were compared with those obtained for muscle using this approach. Serum and muscle samples from enrofloxacin-dosed chickens were also analyzed with this method. The results show that enrofloxacin can be determined in both serum and muscle of chickens dosed at a level formerly approved by the U.S. Food and Drug Administration, for up to at least 48 h after withdrawal from dosing, and suggest that serum can provide an efficient matrix for monitoring FQ levels in chicken.