High-pressure Liquid Chromatography Column Research Articles

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Detection of Benzo[a]pyrene Diol Epoxide Adducts to Histidine and Lysine in Serum Albumin In Vivo by High-Resolution-Tandem Mass Spectrometry.

Electrophilic diol epoxide metabolites are involved in the carcinogenicity of benzo[a]pyrene, one of the widely studied polycyclic aromatic hydrocarbons (PAHs). The exposure of humans to this PAH can be assessed by measuring stable blood protein adducts, such as to histidine and lysine in serum albumin, from their reactive metabolites. In this respect, measurement of the adducts originating from the genotoxic (+)-anti-benzo[a]pyrene diol epoxide is of interest. However, these are difficult to measure at such low levels as are expected in humans generally exposed to benzo[a]pyrene from air pollution and the diet. The analytical methods detecting PAH-biomarkers still suffer from low selectivity and/or detectability to enable generation of data for calculation of in vivo doses of specific stereoisomers, for evaluation of risk factors and assessing risk from exposures to PAH. Here, we suggest an analytical methodology based on high-pressure liquid chromatography (HPLC) coupled to high-resolution tandem mass spectrometry (MS) to lower the detection limits as well as to increase the selectivity with improvements in both chromatographic separation and mass determination. Method development was performed using serum albumin alkylated in vitro by benzo[a]pyrene diol epoxide isomers. The (+)-anti-benzo[a]pyrene diol epoxide adducts could be chromatographically resolved by using an HPLC column with a pentafluorophenyl stationary phase. Interferences were further diminished by the high mass accuracy and resolving power of Orbitrap MS. The achieved method detection limit for the (+)-anti-benzo[a]pyrene diol epoxide adduct to histidine was approximately 4 amol/mg serum albumin. This adduct as well as the adducts to histidine from (−)-anti- and (+/−)-syn-benzo[a]pyrene diol epoxide were quantified in the samples from benzo[a]pyrene-exposed mice. Corresponding adducts to lysine were also quantified. In human serum albumin, the anti-benzo[a]pyrene diol epoxide adducts to histidine were detected in only two out of twelve samples and at a level of approximately 0.1 fmol/mg.

Chiral organic stereochemistry: Chiral HPLC, chiral auxiliaries, CD spectroscopy, X-ray crystallography, and light-powered chiral molecular motors.

In the studies of chiral organic stereochemistry, it is important to use enantiopure compounds. For this purpose, the chiral HPLC (High-Pressure Liquid Chromatography) columns containing chiral stationary phases were invented by Y. Okamoto and coworkers for enantio-separating various racemic compounds. In addition, the use of chiral auxiliaries is also useful for preparing enantiopure compounds and also for determining their absolute configurations, where covalent-bonded diastereomers are separated by HPLC on silica gel. In this review article, these HPLC methods will be discussed together with the applications to some interesting organic compounds including light-powered chiral molecular motors.

Tetrodotoxin and Its Analogs: A Review of Analysis Methods and Levels in Pufferfish

Pufferfish is a major source of tetrodotoxin (TTX), a potent neurotoxin that can lead to death in humans if consumed. The content of TTX and its analogs in different pufferfish species and the distribution in various fish organs are poorly understood. In this study, we compared TTX analysis methods, including mouse bioassay, enzyme-linked immunosorbent assay, high-pressure liquid chromatography (HPLC) coupled with a fluorescence detector, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry (LC-MS) methods. Different HPLC columns (e.g., amide, hydrophilic interaction liquid chromatography, and C18 columns) and mobile phases were compared for TTX analysis using LC-MS. Additionally, published extraction methods were compared for LC-MS analysis. The content of TTX and its analogs in different pufferfish species and their distribution in various organs (e.g., liver, muscle, intestine, testis, ovary, and skin) were summarized. This information can help understand the available TTX analysis methods, variation in TTX contents in pufferfish species from different regions, and TTX distribution in various organs.

Improved Sensitivity in Low-Input Proteomics Using Micropillar Array-Based Chromatography.

Capitalizing on the massive increase in sample concentrations which are produced by extremely low elution volumes, nanoliquid chromatography–electrospray ionization-tandem mass spectrometry (nano-LC–ESI-MS/MS) is currently one of the most sensitive analytical technologies for the comprehensive characterization of complex protein samples. However, despite tremendous technological improvements made in the production and the packing of monodisperse spherical particles for nanoflow high-pressure liquid chromatography (HPLC), current state-of-the-art systems still suffer from limits in operation at the maximum potential of the technology. With the recent introduction of the μPAC system, which provides perfectly ordered micropillar array based chromatographic support materials, completely new chromatographic concepts for optimization toward the needs of ultrasensitive proteomics become available. Here we report on a series of benchmarking experiments comparing the performance of a commercially available 50 cm micropillar array column to a widely used nanoflow HPLC column for the proteomics analysis of 10 ng of tryptic HeLa cell digest. Comparative analysis of LC–MS/MS-data corroborated that micropillar array cartridges provide outstanding chromatographic performance, excellent retention time stability, and increased sensitivity in the analysis of low-input proteomics samples and thus repeatedly yielded almost twice as many unique peptide and unique protein group identifications when compared to conventional nanoflow HPLC columns.

Development of comprehensive two-dimensional liquid chromatography for investigating aging of plastic bonded explosives

The feasibility of measuring the aging and degradation of PBX 9501 via online two dimensional liquid chromatography (LC × LC) is investigated, and a preliminary instrumental setup and method is developed. Plastic-Bonded eXplosive (PBX) 9501 is nominally composed of 94.9 wt% HMX, 2.5 wt% Estane® 5703 (poly (ester urethane)), 2.5 wt% BDNPA/F (nitroplasticizer), 0.1 wt% Irganox 1010 and PBNA (N-phenyl-naphthylamine) at low concentrations. When exposed to various environmental conditions, PBX 9501 will degrade through different pathways. Because PBX 9501 is composed of both low molecular weight compounds (BDNPA/F, Irganox 1010, PBNA, and potential degradation products) and high molecular weight compounds (Estane® 5703), analysis is normally performed via two independent analyses. The low molecular weight species are analyzed via high pressure liquid chromatography (HPLC) and the high molecular weight species via size exclusion chromatography (SEC). While these individual techniques yield information about the aging of PBX 9501, the combination of HPLC and SEC (i.e. HPLC × SEC) can simplify and streamline the analyses while also providing additional chemical information. A simplified sample preparation method is proposed for LC × LC analysis. Various SEC columns and HPLC column selection, flow rate, and gradient ramps were investigated for their application of measuring aged PBX 9501. Finally, two LC × LC separations of a library standard of PBX 9501 and a sample of aged PBX 9501 are compared.

Polymerized high internal phase emulsion monoliths for the chromatographic separation of engineered nanoparticles

ABSTRACTPolyHIPEs of ethylene glycol dimethacrylate (EGDMA) and styrene/divinylbenzene were prepared by polymerization of water‐in‐oil high internal phase emulsions (HIPEs) within high pressure liquid chromatography (HPLC) columns. The columns were incorporated into a HPLC system affixed to an inductively‐coupled plasma mass spectrometer, and their potential for the separation of engineered nanoparticles was investigated. Triplicate injections of 5 and 10 nm gold particles injected onto a poly(styrene‐co‐divinylbenzene) polyHIPE column produced an average difference in retention time of 135 s. On a poly(EGDMA) column, triplicate injections of dysprosium containing polystyrene particles of 52 and 155 nm produced a difference in retention time of 8 s. In both cases the smaller particles eluted from the column first. Comparison, using scanning electron microscopy, of the polyHIPE columns after the separations, against freestanding monoliths produced from the same HIPEs, revealed no apparent change in the internal porous structure of the polyHIPEs. © 2015 The Authors Journal of Applied Polymer Science Published by Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 132, 41229.

Evaluating the interactions of organic compounds with multi-walled carbon nanotubes by self-packed HPLC column and linear solvation energy relationship

Understanding the interactions between organic pollutants and carbon nanotubes (CNTs) is critical for fate assessment of both CNTs and organic pollutants. In this study, the chromatographic approach was introduced based on CNTs as stationary phase for the evaluation of such interactions. The pristine multi-walled carbon nanotubes (MWCNTs) were packed into columns of high-pressure liquid chromatography (HPLC) and the retention factors (k′) were determined to characterize the adsorption affinity of organic compounds onto MWCNTs. Nine compounds were tested. The results showed that their lnk′ values followed the order: benzene<toluene<phenol<chlorobenzene<bromobenzene<aniline<sulfamethoxazole<sulfadiazine≈sulfadimidine. The linear solvation energy relationship (LSER) theory was adopted to correlate lnk′ with the molecular solvatochromic parameters. We found that lnk′ of the studied compounds correlate positively with molecular polarizability (E) significantly, suggesting that the π-/n-electrons-dependent polarizable interactions play a major role for the adsorption. Moreover, the thermodynamic parameters calculated from van’t Hoff equations revealed that the interactions between the compounds and MWCNTs were spontaneous and exothermic processes.

Quantitation of leukotriene B4 in human sputum as a biomarker using UPLC–MS/MS

Leukotriene B4 (LTB4) is a potent mediator of inflammation and has been recognized as an important target for therapeutic intervention for treatment of diseases such as asthma. In the current work, a highly selective and sensitive UPLC-MS/MS assay was developed for quantitation of LTB4 in human sputum as a biomarker for LTB4 biosynthesis inhibition. A fit-for-purpose strategy for method development, assay qualification, and study support was adopted for this biomarker project. A surrogate matrix (protein buffer) was used for preparation of calibration samples and certain levels of quality control (QC) samples to avoid interference from endogenous analyte, while the low QC was prepared in authentic matrix, human sputum. The analytical methodology utilized a liquid-liquid extraction procedure in 96-well plate format. Chromatographic separation was achieved with a reversed-phase ultra high pressure liquid chromatography (UPLC) column using gradient elution, and the run time was 4.5min per sample. The lower limit of quantitation (LLOQ) was 0.2ng/mL, and the calibration curve range was 0.2-20ng/mL. Acceptable accuracy, precision, linearity, specificity, recovery, and matrix effect was obtained. Bench-top stability (6h), freeze-thaw stability (3 cycles at -20°C), and autosampler stability (97h at ambient temperature) all met acceptance criteria. Frozen long-term stability for 166 days at -20°C in sputum did not meet acceptance criteria by showing only ≥75% of nominal concentration and the information was taken into consideration for study support. Two important observations in the current work were: (1) LTB4 was unstable in sputum in the presence of liquification reagent dithiothreitol (DTT). Therefore, a non-DTT treatment method for sputum processing was developed and applied to the bioanalytical assay and clinical study support; and (2) chromatographic separation of LTB4 from its three non-enzymatically derived isomers, i.e. 6-trans-LTB4, 12-epi-LTB4, and 6-trans-12-epi-LTB4, was achieved. This assay was successfully applied to a Phase II clinical study for proof-of-concept of a LTA4 hydrolase inhibitor for treatment of asthma.

Perspectives on the Evolution of the Column Efficiency in Liquid Chromatography

When analyses of mixtures of small molecules are carried out at mobile phase velocities close to (for isocratic runs) or somewhat above (for gradient runs) the optimum velocity, the eddy diffusion term contributes to at least 75% of the band broadening. Future improvements in column performance may come only from a reduction of the eddy diffusion term. The classical models of axial dispersion of Gunn and Giddings are revisited and their predictions compared to recently reported eddy dispersion data obtained by solving numerically the Navier-Stokes equations and simulating advective-diffusive transport in the bulk region and in confined geometries of reconstructed and computer-generated random sphere packings. The Gunn model fails to describe these data. In contrast, the Giddings model succeeds, provided that his original guesses regarding the values of two parameters of his model are adjusted. Accurate measurements of real eddy dispersion data in modern high-pressure liquid chromatography (HPLC) columns were performed by applying a well established experimental protocol. Their results demonstrate that the other contribution to band broadening, sample dispersion in the homogeneous bulk region of these packed beds, accounts for less than 30% of the total eddy dispersion at velocities larger than the optimum velocity. This shows that the resolution power of modern HPLC columns is essentially controlled by wall and/or border layer trans-column eddy dispersion effects, depending on whether the column is radially equilibrated or not. Under a preasymptotic dispersion regime, the performance of short and wide HPLC columns is controlled by the border effects. As the bed aspect ratio (D/dp) increases, the column performance tends toward that of the infinite diameter column. Further improvement appears possible using radial segmentation of the outlet flow. Under an asymptotic dispersion regime, the reduced column plate height of long and thin HPLC columns is controlled by the wall effects and can be optimized only by improving the packing procedures, keeping as low as possible the bed aspect ratio and maximizing the transverse dispersion coefficient.

Modelling interactions between a PBB and fullerenes

Fullerenes have many uses including in medical and electronic nanodevices. High pressure liquid chromatography (HPLC) columns are generally used to extract a certain structure of fullerne from a mixture of them. In this paper, we investigate the interactions between various types of fullerenes and a station phase in HPLC known as pentabromobenzyl (PBB). The Lennard-Jones potential and a continuum approach are employed to determine the van der Waals energy of these interactions within the HPLC columns. The equilibrium configurations for any given distance between a fullerene and the centre of a PBB are obtained. Results of this study may assist the design of a chromatography column for fullerene separation.

Development of Budesonide Nanocluster Dry Powder Aerosols: Formulation and Stability

The physical and chemical stability of dry powder aerosol formulations is an essential component in the development of an inhaled therapeutic. The pharmaceutical processing methods and storage conditions are primary determinants of the stability of a dry powder inhaler (DPI) formulation. Wet milling was used to produce budesonide NanoClusters (NCs), which are agglomerates of drug nanoparticles (≈ 300 nm) with a mean aerodynamic diameter between 1 and 3 µm, capable of deep lung penetration. In this study, the reproducibility of NC processing and performance was established. The physical stability of a selected budesonide NC formulation was investigated using industry standard dose content uniformity and cascade impaction techniques. The chemical stability of the lead formulation was also determined as a function of processing parameters and storage conditions. This study confirms the reproducibility and robust stability of NC powders as a novel means to turn drug particles into high-performance aerosols.

Aflatoxin B1 in post-harvest peanuts and dietary risk in China

To monitor the aflatoxin contamination status in raw peanuts and evaluate the effect on public health, 1040 samples were collected from four agro-ecological zones throughout 12 provinces from 2009 to 2010 in China and then analyzed for aflatoxin B1 (AFB1) levels using High Pressure Liquid Chromatography (HPLC) and immunoaffinity columns. The results revealed that AFB1 was detected in 25% of the samples, ranging from 0.01 to 720 μg/kg. The Monte Carlo and bootstrap methods were employed to estimate AFB1 intake in children and adults and their potential liver cancer risk. The mean estimated intakes for children and adults were 0.218–0.222 ng/kg body weight (bw)/day and 0.106–0.108 ng/kg bw/day. The liver cancer risk, calculated by two approaches derived from the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and European Food Safety Authority (EFSA), were estimated at 0.003–0.17 cancer cases/year/100,000 and 24.7–1273 margins of exposure values, respectively. The results suggest that AFB1 contamination in raw peanuts and dietary risk was low, but essential surveillance measures should be taken to protect public health.

High‐throughput analysis using gated multi‐inlet mass spectrometry

Gated multi-inlet mass spectrometry is introduced for high-throughput chemical analysis. In this design, multiple high-pressure liquid chromatography (HPLC) columns or capillary electrophoresis (CE) capillaries are attached to multiple electrosprayers (one for each column or capillary) that spray toward a gated multi-inlet time-of-flight mass spectrometer (TOF-MS). Although all of the sprayers are spraying continuously, only one inlet is exposed at any given time for a specific duration set by the MS data system. The gated multi-sprayer, multi-inlet design significantly enhances the performance of the multi-ESI, multi-inlet TOF-MS with minimal cost and reduces analysis time. The gated multi-sprayer, multi-inlet design was applied to the investigation of column-to-column reproducibility of multiple HPLCs using a peptide mixture and to the simultaneous analysis of four protein digests. In addition, it was applied to the analysis of peptide mixtures using eight CE capillaries. The gated multi-inlet MS has several advantages compared to our previous non-gated multi-inlet MS. For example, because only one inlet is open at one time, the original manufacturer's inlet inner diameter and pumping system can be used, which enhances the sensitivity of detection for each inlet and minimizes the manufacturing cost. In addition, the number of inlets can be increased as desired. The maximum number of liquid streams that can be concurrently analyzed is limited by: (1) the number of inlets, (2) the chromatographic (electrophoretic) peak width, and (3) how fast the gate can move from one position to the next.

The Ubiquitin Ligase Nedd4-1 Is Required for Heart Development and Is a Suppressor of Thrombospondin-1

Nedd4 (Nedd4-1) is a Hect domain E3 ubiquitin ligase that also contains a C2 domain and three WW domains. Despite numerous in vitro studies, its biological function in vivo is not well understood. Here we show that disruption of Nedd4-1 in mice (leaving Nedd4-2 intact) caused embryonic lethality at mid gestation, with pronounced heart defects (double-outlet right ventricle and atrioventricular cushion defects) and vasculature abnormalities. Quantitative mass spectrometry and immunoblot analyses of lysates from the wild type and knock-out mouse embryonic fibroblasts to identify Nedd4-1 in vivo targets revealed dramatically increased amounts of thrombospondin-1 (Tsp-1) in the knock-out mouse embryonic fibroblasts and embryos. Tsp-1 is an inhibitor of angiogenesis, and its elevated level was mediated primarily by enhanced transcription. Interestingly, the administration of aspirin (an inhibitor of Tsp-1) to the pregnant heterozygote mothers led to a reduction in Tsp-1 levels and a substantial rescue of the embryonic lethality. These results suggest that Nedd4-1 is a suppressor of Tsp1 and that increased levels of Tsp-1 in the Nedd4-1 knock-out mice may have contributed to the developmental defect observed in the embryos.

Recognition Versus Adaptive Up-regulation and Degradation of CC Chemokines by the Chemokine Decoy Receptor D6 Are Determined by Their N-terminal Sequence

The chemokine decoy receptor D6 controls inflammatory responses by selective recognition and degradation of most CCR1 to CCR5 agonistic ligands. CCL14 is a homeostatic chemokine present at high concentrations in the serum with a weak agonist activity on CCR1. Under inflammatory conditions, plasmin and UPA-mediated truncation of 8 amino acids generates the potent CCR1/CCR3/CCR5 isoform CCL14(9-74), which is further processed and inactivated by dipeptidyl peptidase IV/CD26 that generates CCL14(11-74). Here we report that D6 efficiently binds both CCL14 and its truncated isoforms. Like other D6 ligands, the biologically active CCL14(9-74) induces adaptive up-regulation of D6 expression on the cell membrane and is rapidly and efficiently degraded. In contrast, the D6-mediated degradation of the biologically inactive isoforms CCL14(1-74) and CCL14(11-74) is very inefficient. Thus, D6 cooperates with CD26 in the negative regulation of CCL14 by the selective degradation of its biologically active isoform. Analysis of a panel of CC chemokines and their truncated isoforms revealed that D6-mediated chemokine degradation does not correlate with binding affinity. Conversely, degradation efficiency is positively correlated with D6 adaptive up-regulation. Sequence analysis indicated that a proline residue in position 2 of D6 ligands is dispensable for binding but crucial for D6 adaptive up-regulation and efficient degradation.

Lipidomic Analysis of Dynamic Eicosanoid Responses during the Induction and Resolution of Lyme Arthritis

Eicosanoids and other bioactive lipid mediators are indispensable regulators of biological processes, as demonstrated by the numerous inflammatory diseases resulting from their dysregulation, including cancer, hyperalgesia, atherosclerosis, and arthritis. Despite their importance, a robust strategy comparable with gene or protein array technology for comprehensively analyzing the eicosanoid metabolome has not been forthcoming. We have developed liquid chromatography-tandem mass spectrometry methodology that quantitatively and comprehensively analyzes the eicosanoid metabolome and utilized this approach to characterize eicosanoid production during experimental Lyme arthritis in mice infected with the bacterium Borrelia burgdorferi. Eicosanoids were extracted throughout infection from the joints of Lyme arthritis-resistant and -susceptible mice and subjected to lipidomic profiling. We identified temporal and quantitative differences between these mouse strains in the production of eicosanoids, which correlated with differences in arthritis development. The eicosanoid biosynthetic enzyme cyclooxygenase (COX)-2 has been implicated in the regulation of Lyme arthritis pathology, and subsequent lipidomic profiling of B. burgdorferi-infected COX-2(-/-) mice identified reductions not only in COX-2 products but, surprisingly, also significant off-target reductions in 5-lipoxygenase metabolites. Our results demonstrate the utility of a comprehensive lipidomic approach for identifying potential contributors to disease pathology and may facilitate the development of more precisely targeted treatment strategies.

A medical device/drug delivery system for treatment of glaucoma

Background: The aim of this study was to investigate the uptake and release kinetics of two common glaucoma drugs delivered onto hydrogel contact lenses using analytical chemistry and to evaluate this device's ability to control intraocular pressure in a limited number of volunteers.Methods: Contact lenses were incubated in a source solution containing timolol maleate or brimonidine tartrate to determine uptake kinetics. The lenses were then immersed in fresh saline to determine release kinetics. Analysis was performed by high‐pressure liquid chromatography (HPLC). HPLC column retention times were determined for drugs released from the lens individually and under co‐elution conditions. To evaluate clinical feasibility and toxicity, three volunteers (patients being treated for glaucoma) were provided with contact lenses that had been passively impregnated with either drug. After a three‐week wash‐out period, the volunteers were instructed to wear the lenses for 30-minutes per day for two weeks.Results: HPLC analysis showed that maximum uptake and release of both drugs had occurred by approximately 60-minutes, with the slopes tending to flatten after this point. The retention time on the HPLC column was 8.08-minutes for timolol maleate and 2.16-minutes for brimonidine tartrate after incubation for one hour, with no changes after seven hours. Patient data showed that use of the lenses maintained IOP at levels equivalent to those obtained with previous treatment. No ocular toxicity was observed.Conclusion: Drugs commonly used for glaucoma treatment can be passively transferred to a hydrogel contact lens and then eluted from the polymer. Data obtained from a limited number of patients suggest that this contact lens/drug delivery system may be a feasible means of controlling IOP.

O-Linked β-N-Acetylglucosaminyltransferase Substrate Specificity Is Regulated by Myosin Phosphatase Targeting and Other Interacting Proteins

O-GlcNAc-transferase (OGT) substrate specificity is regulated by transiently interacting proteins. To further examine the regulation of OGT, we have identified 27 putative OGT-interacting proteins through a yeast two-hybrid screen. Two of these proteins, Trak1 (OIP106) and O-GlcNAcase, have been shown previously to interact with and regulate OGT. We demonstrate here that MYPT1 and CARM1 also interact with and target OGT. MYPT1 and CARM1 are substrates of OGT in vitro and in vivo. MYPT1 and CARM1 also function to alter OGT substrate specificity in vitro. Furthermore depletion of MYPT1 in Neuro-2a neuroblastoma cells alters GlcNAcylation of several proteins under basal conditions, suggesting that MYPT1 regulates OGT substrate specificity in vivo.

High performance liquid chromatographic determination of plasma free and total tazobactam and piperacillin

A high-pressure liquid chromatography (HPLC) method with ultraviolet detection was developed for the measurement of plasma free and total tazobactam and piperacillin. This method is simple and fast, requiring only 11 min for the HPLC run and a sample preparation of about 11 min for total drugs and 10 min for free drugs. The procedure for the assay involves the treatment of plasma with acetonitrile for total drugs determination, and the use of a centrifugal filter device to deproteinize plasma for free drugs determination. The HPLC column, a Hypersil-ODS, was equilibrated with an eluent mixture composed of acetonitrile–potassium phosphate (pH 2.6). CVs for repeatability of tazobactam and piperacillin measurements ranged from 4.30 to 6.60; CVs for reproducibility ranged from 5.60 to 9.40. Mean analytical recoveries ranged from 100.4 to 103%. A linear relationship was obtained between peak area and drugs concentration in the range studied (0–62.5 mg/L for tazobactam and 0–500 mg/L for piperacillin). The equation for regression line were y = 19 x − 1.4 for tazobactam and y = 1.7 x − 0.9 for piperacillin; correlation coefficients were >0.999. The lower limit of quantitation (LLQ) for standard samples was about 0.12 mg/L for tazobactam and 0.49 mg/L for piperacillin, respectively. The lower limit of detection (LLD) was 0.06 mg/L for tazobactam and 0.24 mg/L for piperacillin. This HPLC assay for tazobactam and piperacillin is sensitive and accurate, and provides a reliable determination of both free and total tazobactam and piperacillin in human plasma, thus allowing the determination of these analytes in patients receiving tazocillin therapy.