Peak Area Integration Research Articles

Enhanced pharmaceutical analysis by CE using dynamic surface coating system

The poor repeatability of migration times in capillary electrophoresis (CE) within an injection sequence and between capillaries can be a difficulty when implementing CE for routine pharmaceutical analysis. The use of a dynamic surface coating has been shown to improve the routine performance of CE. The surface coating generates an appreciable electro-osmotic flow at low pH, which reduces analysis times for basic drugs compared to the low pH buffers typically used in CE. The repeatability of migration times and repeatability of migration times between capillaries was improved. Peak tailing for basic drugs was also reduced which improved peak shapes and peak area integration precision. It is concluded that the dynamic coating system is a positive advance in the routine implementation of CE into pharmaceutical analysis.

Quantitation in gradient high performance liquid chromatography/inductively coupled mass spectrometry investigated using diclofenac and chlorpromazine.

The use of directly coupled high performance liquid chromatography/inductively coupled plasma mass spectroscopy (HPLC/ICPMS) employing chlorine ((35)Cl/(37)Cl) detection has been investigated with respect to the detection and quantitation of the drugs diclofenac and chlorpromazine. By integration of peak areas in the 'chloratogram' (the chlorine specific HPLC chromatogram), a calibration curve was constructed, from which the concentrations could be determined. Chlorine detected HPLC/ICPMS is quantitative over a wide range of concentrations of pharmaceutical relevance for metabolite detection and the results reproducible (standard deviation +/- 0.43%) over multiple injections. Application of gradient chromatography and variation in the bulk mobile phase physicochemical properties has little effect on the ICPMS detection response for these compounds. This work indicates that the use of HPLC/ICPMS is likely to be quantitatively reliable for metabolism studies for a range of chlorinated xenobiotics.

Simultaneous determination of mepivacaine, tetracaine, and p-butylaminobenzoic acid by high-performance liquid chromatography

Introduction: The purpose of the present study was to develop a simple method for the simultaneous determination of mepivacaine, tetracaine, and p-butylaminobenzoic acid (BABA) in human plasma using high-performance liquid chromatography (HPLC) with a multiwavelength detector. Methods: Human blood samples containing heparin, as an anticoagulant, and physostigmine (100 μg/ml), as an anticholinesterase, or human plasma containing physostigmine were spiked with various concentrations of mepivacaine, tetracaine and, in some cases, BABA. Blood samples were centrifuged and plasma was deproteinized with trifluoroacetic acid (TFA; 7%). After centrifugation, the pH was adjusted to 4.5 and an aliquot of 20, 50 or 100 μl was injected into the HPLC apparatus. The detection was done simultaneously at wavelengths of 214 and 300 nm. Analytical chromatography was done on a Waters μBondapak C 18 reverse-phase column (3.9×300 mm; particle size 10 μm) with a 30-min increasing linear gradient of 20–40% acetonitrile+0.05% TFA in H 2O+0.05% TFA at a flow rate of 1 ml/min. The Waters HPLC system included two pumps, an automatic injector, a column oven, and a M490 multiwavelength detector. Quantification was done using integration of peak areas with peaks of authentic mepivacaine, tetracaine, and BABA standards. Results: Calibration curves for standards of mepivacaine, tetracaine, and BABA were linear in the concentration ranges from 0.1 to 100 μg/ml, and the correlation coefficients exceeded .99 for all compounds. The lower limits of detection were 100 ng/ml for mepivacaine and 50 ng/ml for tetracaine and BABA. The yields for mepivacaine, tetracaine, and BABA were 91±2.1%, 82±3.3%, and 88±2.0% (mean±S.E.M., n=6), respectively. Degradation of tetracaine by human plasma at 37 °C was inhibited by physostigmine. Discussion: The method is sensitive enough to allow blood concentration determinations of mepivacaine and tetracaine or its metabolite, BABA, following local anesthesia induced by these two drugs, especially when their toxic effect may be present. This method also may be useful in forensic medicine for determination of the cause of death after local anesthesia with mepivacaine and/or tetracaine.

Archaeal community structures in rice soils from different geographical regions before and after initiation of methane production

The methane production potential of rice soils, which are situated in different geographical regions, shows inherent variations and is catalyzed by archaeal methanogens. We therefore investigated the archaeal community structure in 11 rice field soils which represent a range of climatic conditions (temperate to subtropical zones) and soil properties. Retrieval of environmental partial SSU rDNA sequences from the rice soils of Shenyang (China) and Gapan (The Philippines) showed that the communities were different from each other. However, despite the differences in soil properties and geographical region the sequences clustered in similar phylogenetic groups to those obtained earlier from rice fields of Vercelli (Italy). The archaeal community structure in the other rice field soils was compared using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the SSU rRNA gene and the methyl-coenzyme M reductase α-subunit gene (mcrA). The relative abundance of each terminal restriction fragment (T-RF) was determined by fluorescence peak area integration. The 182-bp SSU rDNA T-RF (representing members of Methanosarcinaceae and rice cluster (RC) VI) was dominant (40–80% contribution) in Chinese soils (Zhenjiang, Changchun, Jurong, Beiyuan, Shenyang) and the Philippine soil of Gapan. The other Philippine soils (Luisiana, Guangzhou, Pila) and the Italian soils (Vercelli, Pavia) showed a dominant 389-bp T-RF (35–40% contribution), representing mainly the novel methanogenic RC-I. All the other T-RF (80, 88, 280, 375 and >800 bp) contributed <20%. Prolonged anoxic incubation (30–200 days) of the air-dried soils resulted in the production of CH4, which was in some soils preceded by a characteristic halt phase. T-RFLP analysis revealed that the soils with a methanogenic halt phase also showed dramatic archaeal population dynamics which were related to the length of the halt phase. Our results show that the archaeal communities in rice field soils of different geographical origin are highly related, but nevertheless exhibit individual patterns and dynamics, thus providing evidence for the active participation of the community members in energy and carbon flow.

Archaeal community structures in rice soils from different geographical regions before and after initiation of methane production

The methane production potential of rice soils, which are situated in different geographical regions, shows inherent variations and is catalyzed by archaeal methanogens. We therefore investigated the archaeal community structure in 11 rice field soils which represent a range of climatic conditions (temperate to subtropical zones) and soil properties. Retrieval of environmental partial SSU rDNA sequences from the rice soils of Shenyang (China) and Gapan (The Philippines) showed that the communities were different from each other. However, despite the differences in soil properties and geographical region the sequences clustered in similar phylogenetic groups to those obtained earlier from rice fields of Vercelli (Italy). The archaeal community structure in the other rice field soils was compared using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the SSU rRNA gene and the methyl-coenzyme M reductase α-subunit gene ( mcrA). The relative abundance of each terminal restriction fragment (T-RF) was determined by fluorescence peak area integration. The 182-bp SSU rDNA T-RF (representing members of Methanosarcinaceae and rice cluster (RC) VI) was dominant (40–80% contribution) in Chinese soils (Zhenjiang, Changchun, Jurong, Beiyuan, Shenyang) and the Philippine soil of Gapan. The other Philippine soils (Luisiana, Guangzhou, Pila) and the Italian soils (Vercelli, Pavia) showed a dominant 389-bp T-RF (35–40% contribution), representing mainly the novel methanogenic RC-I. All the other T-RF (80, 88, 280, 375 and >800 bp) contributed <20%. Prolonged anoxic incubation (30–200 days) of the air-dried soils resulted in the production of CH 4, which was in some soils preceded by a characteristic halt phase. T-RFLP analysis revealed that the soils with a methanogenic halt phase also showed dramatic archaeal population dynamics which were related to the length of the halt phase. Our results show that the archaeal communities in rice field soils of different geographical origin are highly related, but nevertheless exhibit individual patterns and dynamics, thus providing evidence for the active participation of the community members in energy and carbon flow.

Antiretrovirals: simultaneous determination of five protease inhibitors and three nonnucleoside transcriptase inhibitors in human plasma by a rapid high-performance liquid chromatography--mass spectrometry assay.

An analytical technique using liquid chromatography (LC) coupled with electrospray-mass spectrometry (ESI--MS) has been developed for the simultaneous determination of five protease inhibitors (PIs): saquinavir, indinavir, ritonavir, nelfinavir, and amprenavir; and three non-nucleoside reverse transcriptase inhibitors (NNRTIs): nevirapine, delavirdine, and efavirenz, in human plasma. This assay allows the elution and identification of these drugs in a single run (10 minutes) using a linear gradient with water and acetonitrile. The procedure involves liquid--liquid extraction. High-performance liquid chromatography (HPLC) separation was achieved on a C18 reversed-phase column, with a linear gradient elution followed by mass spectrometry detection. The calibration curves, obtained by automatic process peak area integration, show a good linearity in a range of concentrations between 20 and 10,000 ng/mL (40--10,000 ng/mL for efavirenz). The limit of detection was approximately 10 ng/mL for seven drugs (25 ng/mL for efavirenz). The coefficients of variation (CV) were always less than 15% for both intraday and interday precision for each compound. The recovery of the eight drugs ranged from 88.5% to 100%. This novel LC/ESI--MS assay provides an excellent method for simultaneous quantitative monitoring of different components of the highly active antiretroviral treatments (HAARTs) in patients treated simultaneously with PIs and NNRTIs, and it has been successfully applied to therapeutic drug monitoring and pharmacokinetic studies.

Contact Angle and Electrochemical Characterization of Multicomponent Thiophene-Capped Monolayers

We have investigated the wetting behavior and electrochemical response of self-assembled monolayers made from ω-(3-thienyl)-alkyltrichlorosilanes and alkyltrichlorosilanes on indium−tin oxide coated glass slides. Contact angle measurements and cyclic voltammetry show that the properties of the monolayers change as the relative loading of the two components is changed by altering the concentration of the deposition solution. The water contact angles indicate that a fully loaded hydrophobic layer is formed, and both water and hexadecane contact angle measurements show that the tethered thiophenes are at the periphery of the monolayer. However, the hexadecane contact angles also imply that the surface roughness exposes the methylene groups of the monolayer chains. Cyclic voltammograms obtained from mixtures of 11-(3-thienyl)undecyltrichlorosilane and decyltrichlorosilane and mixtures of 16-(3-thienyl)hexadecyltrichlorosilane and hexadecyltrichlorosilane show that as the relative amount of thienyl-bearing chains in the monolayer decreases, the oxidation peak position shifts to higher potential and the peak area, which is related to the amount of charge transferred, decreases. Integration of the oxidation peak area from the cyclic voltammetry experiments yields a coverage that is consistent with a monolayer. Preliminary evidence for formation of a surface-tethered redox-active polymer is observed by cycling the self-assembled monolayers at low potentials.

Energy Dispersive X-Ray (EDX) and Electron Energy-Loss (EELS) Spectroscopic Mapping of Microelectronic Devices

Abstract Many crucial measurements in the semiconductor industry involve determining the root cause of an electrical failure, often requiring the capability of extracting microstructural and chemical information with nanometer resolution [1]. The microanalysis is achieved by stepping the focused probe over the region of interest in STEM mode and recording an EDX and EELS spectrum at each pixel. Even for relatively modest image sizes, the resultant spectrum-image may consist of more than 10,000 spectra. With such large data sets, the prospect of manually inspecting and quantifying each spectrum in real-time is impossible and even automatic background modeling followed by peak area integration over selected spectral regions-of-interest can be excessively time consuming. Other methods to extract useful physical information include “fingerprinting” characteristic ELNES shapes to known compounds or bonding environments, multiple least squares regression to a relatively small set of suspected components or peak-fitting to model systematic changes in electronic density of states.

A computational study on the effect of random noise on the precision and accuracy of the integration of chromatographic peaks

This paper describes the results of a computational study on the effect of random noise on the precision and accuracy of the integration of chromatographic peaks. The focus of the study is to provide guidance for practical integration of peaks in the presence of random noises. The effect of noise is examined for the following integration parameters: noise levels, number of data points, integration range, digitally filtering (bunching), and peak asymmetry. The baseline is determined from the first and last points of the peaks. We conclude that the major contribution to peak area integration imprecision is the effect of noise on the determination of peak baseline. For all the parameters examined, the signal-to-noise ( S/ N) ratio has the most significant effect on the precision. The number of data points within a peak should be about 30 to provide the best integration precision and peak representation. The absolute integration range should be as small as possible, however, for peaks with different asymmetry and width, the precision is determined by the relative integration range if other integration parameters are kept about the same. Moreover, the increase in peak asymmetry results in the decrease in S/ N ratio, and then an increase in the imprecision. Finally, bunching is always preferred to improve the precision. The results of the study should be applicable to situations where random noises are dominant such as in capillary electrophoresis.

Automated frit inlet/frit outlet flow field-flow fractionation for protein characterization with emphasis on polymeric wheat proteins.

A flow field-flow fractionation (FFF) unit fitted with a 254-nm spacer, frit inlet (FI), and frit outlet (FO) was automated for protein analysis by addition of a system controller, autosampler, and computer software to control pumps, sample loading, and data capture. Standard molecular size marker proteins and polymeric wheat storage protein extracts were used to assess the performance of the automated unit. Optimum resolution for these proteins was obtained with a sample inlet flow of 0.2 mL/min, a frit inlet flow (recirculating) of 1.4 mL/min, and a cross-flow (recirculating) of 5 mL/min using 0.05 M acetic acid containing 0.002% FL-70 as a carrier. Use of the FIFO FFF eliminates the requirement for stop-flow relaxation and pressure balancing, results in better reproducibility, and generates a 7-10-fold increase in sensitivity at the detector by concentrating fractions eluting from the channel. These improvements resulted in superior resolution of polymeric wheat protein fractions compared to those obtained previously using a standard channel with manual load and stop-flow relaxation, allowing accurate integration of peak or size range areas. Automation of this system allows unattended sample fractionation and hence markedly increases potential for sample throughput.

Simultaneous analysis of 14 non-steroidal anti-inflammatory drugs in human serum by electrospray ionization—tandem mass spectrometry without chromatography

A method is described for the simultaneous analysis of 14 non-steroidal anti-inflammatory drugs (NSAIDS) in human serum using negative electrospray ionization-tandem mass spectrometry (ESI-MS/MS). After addition of internal standard and protein precipitation using acetonitrile, samples were transferred to autosampler vials for direct analysis without chromatography. Injection of an air bubble with the sample and a multiple reaction monitoring (MRM) method using argon collision-induced dissociation (CID) of analyte (M-H)- ions permitted integration of the product ion peak areas to produce reproducible quantitative data over the range of concentrations expected in serum during routine use of these drugs. The method permitted the analysis of 30 samples per hour. Two hundred fifty consecutive analyses did not adversely affect instrument sensitivity.

Physicochemical Characterization of Liquid Crystalline Phases in Model Bile and Lipid Digestive Mixtures: A 2H Nmr Study

Phase properties of aqueous dispersions of model bile mixtures containing cholesterol, lecithin and bile salts and model mixed lipid digestive mixtures containing cholesterol, fatty acid, monoglyceride, lecithin and bile salts have been characterized by 2H NMR. We have chemically incorporated a deuteriomethyl (CD3) group into one or two lipid components and utilized differences in motionally-averaged quadrupole splittings (Δv) and CD3 chemical shifts, to determine directly by peak-area integration, for a number of compositions on the phase diagram, the distribution of a 2H-labeled component between micellar, lamellar and solid phases, as well as the chemical compositions of these phases. Our data show that increasing cholesterol stabilizes lamellar phases whereas bile salts disrupt them and favor micelle formation.

A systematic approach to the solid-phase synthesis of linear and cyclic pseudopeptide libraries containing psi[CH2NH] amide bond surrogates.

A systematic approach has been adopted for the synthesis and characterization of a series of linear and cyclic pseudopeptide mixtures containing the psi[CH2NH] amide replacement. The parent structures were based on biologically relevant compounds including an enkephalin analog, H-Tyr-D-Ala-Gly-Phe-Leu-OH, and an Arg-Gly-Asp peptide sequence. The linear mixtures containing 4 and 64 pseudopeptide components with 1, 2 or 3 amide bond surrogates were synthesized using Boc-SPPS. The amount of desired linear pseudopeptides in the mixtures ranged from 67 to 90% as determined by integration of HPLC peak areas. Comparative studies indicated: (i) racemization is not a problem in the synthesis of pseudopeptide mixtures containing the psi[CH2NH] surrogate; and (ii) protection of the psi[CH2NH] surrogate with a benzyloxycarbonyl group during the synthesis is beneficial. Cyclic mixtures containing 4 and 256 cyclic pseudopeptide components with a single amide bond surrogate were synthesized using a resin-bound cyclization approach featuring side-chain attachment of Boc-Asp-OFm to the solid support. Cyclization kinetic studies revealed that the newly developed HATU coupling reagent provided a fast cyclization rate for a pseudopeptide mixture and that the position of the reduced peptide bond within a peptide mixture had only a small effect on the cyclization rates of the mixture. Pseudopeptide libraries permit the more efficient bioassay of complex structures and can also be used to reveal more rapidly trends in physicochemical variables. For example, we observed that the expected increase in hydrophilicity with psi[CH2NH] substitutions during RP-HPLC analysis did not continue with several such replacements.

High-Performance Liquid Chromatographic Resolution of NADP+after Induction of Fluorescence and Its Application to Assay for an NADPH-Dependent Enzyme: Application to the Determination of Glutathione Reductase Activity in Plant Leaf Extracts

A method is described for the determination of glutathione reductase activity (GR; EC 1.6.4.2) in plant extracts utilizing HPLC quantitation of NADP+following the reduction of glutathione disulfides. After protein incubation, fluorescence of NADP+was induced under strongly basic conditions, and the product was directly resolved from the reaction medium by isocratic reversed-phase elution on a silica-coated alumina support which took 2 min. The mobile phase was acetonitrile–water (50:50) delivered at a flow rate of 1.5 ml/min. The adduct (stable for at least 7 days) was detected fluorometrically and quantitated by direct integration of peak area.

A channel-by-channel method of reducing the errors associated with peak area integration

Abstract A new method of reducing the errors associated with peak area integration has been developed. This method utilizes the signal content of each channel as an estimate of the overall peak area. These individual estimates can then be weighted according to the precision with which each estimate is known, producing an overall area estimate. Experimental measurements were performed on a small peak sitting on a large background, and the results compared to those obtained from a commercial software program. Results showed a marked decrease in the spread of results around the “true” value (obtained by counting for a long period of time), and a reduction in the statistical uncertainty associated with the peak area.

Role of the leader and structural regions of prelantibiotic peptides as assessed by expressing nisin-subtilin chimeras in Bacillus subtilis 168, and characterization of their physical, chemical, and antimicrobial properties.

Biosynthesis of lantibiotics such as nisin and subtilin involves post-translational modifications, including dehydration of serines and threonines, formation of thioether cross-linkages, translocation, cleavage of a leader sequence, and release into the medium. We have studied the cellular machinery that performs the modifications by constructing and expressing nisin-subtilin chimeric prepeptides in a strain of Bacillus subtilis 168 that possesses all of the cellular machinery for making subtilin except for the presubtilin gene. The chimeras consisted of a normal subtilin leader region (SL), fused to nisin-subtilin chimeric structural regions, one of which was SL-Nis1-11-Sub12-32, in which the N-terminal portion of the structural region was derived from nisin, and the C-terminal portion derived from subtilin. This chimera was accurately and efficiently converted to the corresponding mature lantibiotic, as established by reverse phase high performance liquid chromatography profiles, proton NMR spectroscopy, mass spectral analysis, and biological activity. A succinylated form of the chimera was also produced. Another chimera was in the reverse sense, with subtilin sequence at the N terminus and nisin sequence at the C terminus of the structural region (SL-Sub1-11-Nis12-34). It was processed into a heterogeneous mixture of products, none of which had the characteristics of a correctly processed polypeptide, but did contain a minor component that was active, with a specific activity that considerably exceeded nisin itself. These results, together with results published earlier, establish that processing requires specific recognition between the prelantibiotic peptide and the processing machinery, and in order for the processing to occur correctly, there must be an appropriate combination of the N-terminal part of the leader region and the C-terminal part of the structural region of the prepeptide.

Determination of Lead in Beer Using Zeeman Background-Corrected Graphite Furnace Atomic Absorption Spectrometry

Because of its toxicity and the potential for adverse health effects, lead has attracted widespread interest in its occurrence in the environment, drinking water, and many comestibles, including alcoholic beverages. Its accurate analysis at very low levels in complex matrices such as beer is subject to many difficulties from interferences and demands specialized equipment and techniques. Graphite furnace atomic absorption spectrometry has been successfully used for the analysis of lead in biological samples and has previously been applied to beer. In recent times, the reassessment of lead's human health implications has prompted North American regulatory agencies to lower the acceptable levels of lead contamination in the environment and, particularly, in drinking water. Consequently, this has renewed the requirements for lower detection limits and improved accuracy in analyses. Earlier studies have shown that with modern-day standards for equipment and materials used in beer production and packaging, beers contain only trace levels of lead and generally were below the newly established guidelines for drinking water. There is need, however, for improved accuracy in lead determinations. This method achieves that by combining an appropriate matrix modifier, platform atomization, peak area integration, Zeeman background correction, insertion of a cool-down step before atomization, and the use of the alternate wavelength (283.3 nm) for lead. The method has a limit of detection of 0.87 μg/L and a limit of determination of 2.1 μg/ L.

A new electrothermal vaporization device for direct sampling of ceramic powders for inductively coupled plasma optical emission spectrometry

In spite of many important advantages of electrothermal vaporization of solid samples combined with inductively coupled plasma spectrometry (ETV-ICP-OES) the method has not been exploited to its full potential as a routine analysis mainly due to unsatisfactory reproducibility. The design of the system which transports the analytes into the plasma is recognized as one of the main factors influencing effective transport. Development of a reliable means of combining an ETV device with an ICP would be very useful when direct analysis of trace elements in ceramic powders is needed. This paper describes the construction of an ETV-ICP system for direct analysis of ceramic powders through in situ thermochemical processing. The system is based on in-line electrical resistive heating of a graphite crucible with powdered silicon carbide mixed with a CoF 2 + BaO modifier in a proportion 1 : 1 : 1. The transport path was shortened to 9 mm from the orifice of the crucible to the bottom of a modified injector tube. No material was deposited on the injector tube based on washing the tube and analyzing the solution by ICP-OES. Initial assessment by peak area integration of emisison signals of 5 mg silicon carbide powder shows that the level of detection lies approximately in pg level for Ti, V, Al and Fe. The system has no memory effect. The ETV unit could be used both by ETV-ICP-OES and ETV-ICP-MS.

In vivo 31P MRS: absolute concentrations, signal-to-noise and prior knowledge.

Absolute metabolite concentrations have been estimated for nucleoside triphosphate and P(i) from in vivo 31P MR measurements using ISIS localization in a rat tumour model, and the results have been compared to those obtained from acid extracts of the tumours. The aim of the experiment was to assess the performance of four different spectral analysis techniques used for absolute quantitation. The spectral analysis techniques used were two frequency domain methods (peak area integration and Lorentzian fitting--FITSPEC) and two time domain methods (VARPRO and HLSVD). The spectra were acquired in blocks so that the degradation in performance of the four spectral analysis methods with decreasing signal-to-noise ratio (SNR) could be compared and referenced. This and the inclusion of a sophisticated method incorporating prior knowledge yields a more realistic and comprehensive protocol than previously published comparisons. The results suggest that VARPRO is the method of choice for quantitative analysis of tumour 31P MR spectra, giving the most reliable results at low SNR.

Chemical composition and antimicrobial activity of essential oil of leaf, stem and rhizome of Alpinia speciosa

AbstractEssential oils prepared by hydrodistillation of leaves (L), rhizomes (R), and stems (S) of Alpinia speciosa (J.C.Wendl.) K.Schum. [=Languas speciosa (J.C.Wendl.) Merrill], Family Zingiberaceae, were analysed by capillary gas chromatography coupled to mass spectrometry (CGC‐MS). Individual components of the oil of each organ were identified by their Kováts indices and their mass spectra which were compared with published material or with data of reference compounds. Quantitative determination was carried out based on peak area integration. The study revealed that the oxygenated compounds constitute 41.7%, 52.1% and 42.8% of the oil in L, R and S, respectively. They are composed mainly of terpinen‐4‐ol (17.3%, 20.2% and 16.0%) and 1,8‐cineole (14.4%, 15.9% and 11.5%) in L, R and S, respectively. The hydrocarbons constitute 52.2% (L), 40.9% (R) and 44.3% (S) of the oil, and comprise mainly sabinene 10.1% (L), 9.8% (R) and 7.5% (S) and γ‐terpinene 11.1% (L), 9.3% (R) and 8.2% (S). The essential oils of the three organs exhibited significant antimicrobial activity against certain Gram‐positive and Gram‐negative bacteria as well as Candida albicans.