Feasibility of near-infrared spectroscopy to detect and to quantify adulterants in cow milk.

Cow milk adulteration involves the dilution of milk with a less-expensive component, such as water or whey. Near-infrared spectroscopy (NIRS) was employed to detect the adulterations of milk, non-destructively. Two adulteration types of cow milk with water and whey were prepared, respectively. NIR spectra of milk adulterations and natural milk samples in the region of 1100 - 2500 nm were collected. The classification of milk adulterations and natural milk were conducted by using discriminant partial least squares (DPLS) and soft independent modelling of class analogy (SIMCA) methods. PLS calibration models for the determination of water and whey contents in milk adulteration were also developed, individually. Comparisons of the classification methods, wavelength regions and data pretreatments were investigated, and are reported in this study. This study showed that NIR spectroscopy can be used to detect water or whey adulterants and their contents in milk samples.     

Residue screening for the beta-agonists clenbuterol, salbutamol and cimaterol in urine using enzyme immunoassay and high-performance liquid chromatography.

The antibody for enzyme immunoassay was raised against clenbuterol-diazo-BSA, and salbutamol-carboxymethyl ether-biocytin was used as a label. Procedural blanks from 500 negative urine samples were always less than 0.2 ppb salbutamol or less than 0.02 ppb clenbuterol equivalents, and a residue level of 1 ppb was detected with good reliability. After treatment of veal calves with anabolic dosages, residue levels in urine amounted to 10-200 ppb clenbuterol or salbutamol. beta-Agonists were separated by high-performance liquid chromatography on LiChrospher RP-Select B columns, and acidic methanol-buffer or acetonitrile-buffer mobile phases. Combinations of high-performance liquid chromatography and enzyme immunoassay were used for confirmation.     

Use of solid-phase microextraction to detect and quantify gas-phase dicarbonyls in indoor environments

Solid-phase microextraction (SPME) was evaluated for the detection and quantification of the gas-phase dicarbonyls, glyoxal (GLY) and methylglyoxal (MGLY). Additionally, polydimethylsiloxane (PDMS), polydimethylsiloxane/divinylbenzene (PDMS/DVB), and carbowax/divinylbenzene (CW/DVB) fibers were tested to determine the optimum fiber for detection of these species. GLY and MGLY were derivatized with O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride (PFBHA), extracted with SPME from headspace or bag chamber and then analyzed by GC/MS. The PDMS/DVB SPME fiber for on-fiber derivatization and subsequent sampling for gas-phase methylglyoxal provided the optimum combination of analytical reproducibility and sensitivity. Linearity of the calibration curve was achieved across a range of 11-222 microg/m(3) (4-75 ppb).     

Food safety: screening tests used to detect and quantify GMO proteins

GMO protein content in maize flours or fresh crops can be easily assessed by the enzyme-linked immunosorbent assays (ELISA) and immunochromatographic (lateral flow) strip tests commercially available. Therefore, ELISA can be seen as a useful tool for screening, for control purposes and for traceability implementation. In order to highlight the importance of monitoring GMO protein presence in food products and to investigate the performance of representative ELISA commercial kits, we evaluated three commercial kits by measuring the amount of Cry1Ab/Cry1Ac in IRMM certified reference materials. Presented at AOAC Europe/Eurachem Symposium March 2005, Brussels, Belgium     

Development of an electronic nose to identify and quantify volatile hazardous compounds

A new electronic nose was developed to identify the chemical compound released when a 2.5-L flask was broken inside a 3 m x 3 m x 2.5 m store-room. Flasks of 10 different hazardous compounds were initially present in the room: ammonia, propanone, hexane, acetic acid, toluene, methanol, tetrachloromethane, chloroform, ethanol and dichloromethane. Besides identification, quantification of the compound present in the air was also performed by the electronic nose, in order to evaluate the risk level for room cleaning. An array of six sensors based on coated piezoelectric quartz crystals was used. Although none of the individual sensors was specific for a single compound, an artificial neural network made it possible to identify and quantify the released vapour, among a series of 10 compounds, with six sensors. The neural network could be simplified, and the number of neurons reduced, provided it was used just for the identification task. Quantification could be performed later using the individual calibration of the sensor most sensitive to the identified compound.     

Feasibility of using a surface plasmon resonance-based biosensor to detect and quantify yessotoxin

Yessotoxin (YTX) is a disulfated polyether toxin produced by marine dinoflagellates. Although there is no clear evidence that YTX is toxic to humans, it is a major cause of false positives in DSP toxin detection by mouse bioassay. We developed a new detection and quantification method for yessotoxin using a BiaCore X Surface plasmon resonance (SPR)-based biosensor. The assay is based in the interaction of YTX with phosphodiesterase enzymes (PDE), one of its cellular targets. The injection of several YTX concentrations (3-12 microM) over immobilized PDE I, showed a dose dependent binding signal, which K(obs) (observed rate constant) allowed us to obtain a calibration curve with a linear fit. The detection of yessotoxin using SPR-based biosensor allows the quantification of the toxin with an automated and repetitive method at concentrations in the range of the 1 mg kg(-1) European regulatory limit.     

Quantitative determination of four diterpenoids in Radix Salviae Miltiorrhizae using LC-MS-MS

An HPLC-ESI-MS-MS method has been developed for the quantitative determination of four diterpenoids, dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone IIA in Radix Salviae Miltiorrhizae (RSM, the root of Salvia miltiorrhiza BGE.). The diterpenoids were chromatographically separated on a C18 HPLC column, and the quantification of these diterpenoids was based on the fragments of [M+H]+ under collision-activated conditions and in Selected Reaction Monitoring (SRM) mode. The quantitative method was validated, and the mean recovery rates from fortified samples (n=5) of dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone IIA were 95.0%, 97.2%, 93.1%, and 95.9% with variation coefficient of 6.0%, 4.3%, 3.7%, and 4.2%, respectively. The established method was successfully applied to the quality assessment of seven batches of RSM samples collected from different regions of China.     

Supercritical fluid extraction of clenbuterol from bovine liver tissue.

The development of a supercritical fluid extraction procedure for the extraction of clenbuterol from bovine liver tissue is described. The procedure involves a combination of supercritical fluid extraction with enzyme immunoassay for the determination of clenbuterol residues at the low ppb level. Method validation incorporating inter- and intra-assays was carried out on fortified liver tissue and showed good recoveries and low variations (RSD < 15%) for levels of clenbuterol of 0.5, 2 and 5 ppb. The developed procedure was shown to be successful for the determination of clenbuterol in incurred liver tissue. The effects of organic modifiers and of inherent sample moisture on analyte extractability are also presented.     

Determination of beta-agonists in liver and retina by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the determination of bromobuterol, cimaterol, clenbuterol, clenpenterol, hydroxymethylclenbuterol, isoxsuprine, mabuterol, ractopamine, ritrodrine, salbutamol, terbutaline, and tulobuterol residues in bovine liver and retina is reported. This procedure uses enzymatic digestion, liquid-liquid extraction, and cleanup on Oasis HLB solid-phase extraction cartridges, followed by determination of the residues by LC-tandem quadrupole MS using atmospheric pressure chemical ionization in the positive ion mode. Overall average recoveries ranged from 23 to 76% for liver and 34 to 77% for retina. The mean values for samples fortified at levels between 0.5-2.0 microg/kg (liver) and 5-20 microg/kg (retina) agreed within 98-118% of the spiked levels, with coefficients of variation ranging from 6 to 20%. The decision limits, CCalpha, ranged from 0.1 to 0.3 microg/kg for liver, 1-3 microg/kg for retina, and detection capabilities, CCbeta, from 0.2-0.5 microg/kg for liver and 2-5 microg/kg for retina.     

A novel sorbent for the determination of clenbuterol in bovine liver.

The use of three C18 sorbents in matrix solid phase dispersion (MSPD) for the determination of clenbuterol in bovine liver fortified at 5 ng g-1 is described. MSPD grade C18 sorbents give rise to more efficient blending and packing of the material for subsequent washing and analyte elution in comparison with a non-MSPD grade C18 sorbent. Following enzymatic deconjugation of the liver extracts, radioimmunoassay is used as the method of determination. The mean recovery of clenbuterol with all sorbents is comparable and within the range 86-96% in two intra-assay studies (n = 3). The liver extracts in each case are highly coloured. The variation in recovery is observed to be lowest with MSPD grade C18 (end-capped). This sorbent was used in further studies to evaluate the use of solid phase extraction (SPE), post MSPD, with normal phase aminopropyl or mixed mode cation exchange columns for extract purification. The mean recovery of clenbuterol (n = 4, inter-assay study) following MSPD and normal phase SPE clean-up was 95 +/- 15% and 89 +/- 9% at fortification levels of 1 and 2.5 ng g-1, respectively.     

Development of a peptide mapping procedure to identify and quantify methionine oxidation in recombinant human alpha1-antitrypsin

A peptide mapping procedure was developed to identify and quantify methionine oxidation in recombinant human alpha1-antitrypsin. Due to the protein's complex structural biochemistry, chromatographic analysis of methionine containing digest peptides was a significant challenge. However, by using a combination of mass spectrometry, protein engineering, and high-temperature reversed-phase liquid chromatography, we were able to identify methionine residues that are susceptible to oxidation by hydrogen peroxide. and quantify their reactivity. Our results show that five of the protein's 10 methionine residues are susceptible to oxidation at neutral pH, four of which are localized to the active site region.     

Development of a MALDI-TOF-MS method to identify and quantify butyrylcholinesterase inhibition resulting from exposure to organophosphate and carbamate pesticides

A novel, proteomics based method was developed for the detection, quantification, and categorization of serum butyrylcholinesterase (BChE) inhibitors, including organophosphates (OPs) and carbamates (CBs). This method was based on the MALDI-TOF-MS analysis of the trypsin generated BChE active site peptide (191-SVTLFGESAGAASVSLHLLSPR-212) previously modified by reaction with an OP or CB. The ionization efficiency of OP modified active site peptides by MALDI was greatly improved by adding diammonium citrate to the MALDI matrix, which made the quantification of OP exposure feasible. Excellent linearity (r2 > 0.98) between the normalized abundance ratios (NARs) and OP concentrations or logarithm of carbaryl concentration was obtained. The accuracy of the developed assay was evaluated by comparison of IC50 and IC100 values from the assay with those determined by the Ellman method. Results from this method were comparable with those from the Ellman method. The advantage of the assay was that both the origin and the extent of pesticide exposure can be determined in one analysis. Our MALDI method can provide critical evidence for the pesticide exposure at low BChE inhibition levels even down to 3%, not available with the Ellman method.     

Highly sensitive and selective fluorescence optosensor to detect and quantify benzo[a]pyrene in water samples

We describe here a method for detecting and quantifying the highly carcinogenic polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) in water, based on a flow-trough optical sensor. The technique is fast (response time of 40 s) and simple and at the same time meets the standards of sensitivity and selectivity required by the European Guidelines on Water for Human Consumption. The optosensor is based on the on-line immobilization of BaP on a non-ionic resin (Amberlite XAD-4) solid support in a continuous-flow system. BaP was analyzed in a 15 mM H₂PO₄⁻/HPO₄²⁻ buffer solution with 25% (v/v) 1,4-dioxane at pH 7. Fluorescence intensity was measured at λ_ex/em=392/406 nm. The experimental conditions (reagent phase, pH, type and concentration of buffer solution and organic solvent) and flow-injection values (flow rate and injection volume) were carefully controlled. Under these conditions the optosensor was sensitive to a linear concentration range of between 3.0 and 250.0 ng l⁻¹ with a detection limit of 3.0 ng l⁻¹ and a standard deviation of 1.5% at 150 ng l⁻¹. The optosensor was applied to the quantification of BaP in drinking and waste water samples (95-105% recovery) in presence of the other 15 EPA PAHs at 1000 ng l⁻¹ concentration level. The influence of other fluorescent polycyclic aromatic hydrocarbons and potential interference from ions usually present in water was also evaluated.     

The use of selected ion flow tube mass spectrometry to detect and quantify polyamines in headspace gas and oral air

Polyamines are a class of aliphatic compounds which include putrescine, cadaverine, spermine and spermidine. They are involved in a variety of cellular processes and have been implicated in a number of different pathophysiological mechanisms. Polyamines are volatile compounds having a distinctive odour normally perceived as being unpleasant. The measurement of their abundance has, however, been restricted to compounds present in the aqueous phase. Using selected ion flow tube mass spectrometry (SIFT‐MS) we have shown that the polyamines react with the ions H₃O⁺, NO⁺ and O to form distinctive product ions allowing their levels to be quantified in the vapour phase. The low volatility of spermine did not allow extensive analysis of this compound by SIFT‐MS while the adherent properties of cadaverine and putrescine required the use of PTFE transfer lines and couplers. Our data suggested the presence of cadaverine and putrescine in both oral air and the headspace of putrefying bovine muscle, while product ions corresponding to putrescine and spermidine were found in the headspace of human semen. SIFT‐MS therefore appears to be a practical means of measuring vapour‐phase polyamine levels, having applications in biology, medicine and dentistry, and food science. Copyright © 2009 John Wiley & Sons, Ltd.     

Use of spatiotemporal response information from sorption-based sensor arrays to identify and quantify the composition of analyte mixtures

Linear sensor arrays made from small molecule/carbon black composite chemiresistors placed in a low-headspace volume chamber, with vapor delivered at low flow rates, allowed for the extraction of new chemical information that significantly increased the ability of the sensor arrays to identify vapor mixture components and to quantify their concentrations. Each sensor sorbed vapors from the gas stream and, thereby, as in gas chromatography, separated species having high vapor pressures from species having low vapor pressures. Instead of producing only equilibrium-based sensor responses that were representative of the thermodynamic equilibrium partitioning of analyte between each sensor and the initial vapor phase, the sensor responses varied depending on the position of the sensor in the chamber and the time since the beginning of the analyte exposure. The concomitant spatiotemporal (ST) sensor array response therefore provided information that was a function of time, as well as of the position of the sensor in the chamber. The responses to pure analytes and to multicomponent analyte mixtures comprised of hexane, decane, ethyl acetate, chlorobenzene, ethanol, and/or butanol were recorded along each of the sensor arrays. Use of a non-negative least-squares (NNLS) method for analysis of the ST data enabled the correct identification and quantification of the composition of two-, three-, four-, and five-component mixtures from arrays using only four chemically different sorbent films. In contrast, when traditional time- and position-independent sensor response information was used, these same mixtures could not be identified or quantified robustly. The work has also demonstrated that, for ST data, NNLS yielded significantly better results than analyses using extended disjoint principal components modeling. The ability to correctly identify and quantify constituent components of vapor mixtures through the use of such ST information significantly expands the capabilities of such broadly cross-reactive arrays of sensors.     

Using second-order calibration to identify and quantify aromatic sulfonates in water by high-performance liquid chromatography in the presence of coeluting interferences

We used the Generalized Rank Annihilation Method (GRAM), a second-order calibration method, to quantify aromatic sulfonates in water with high-performance liquid chromatography (HPLC) when interferences coeluted with the analytes of interest. With GRAM, we can quantify in only two chromatographic analyses, one for a calibration sample and one for the unknown sample. The calculated concentrations were not statistically different to those obtained when the chromatographic separation of the unknown sample was modified in order to completely separate the analyte from the interferences before univariate calibration. With GRAM, the concentrations are determined much more quickly because a complete resolution is not required.     

Pharmacokinetics of clenbuterol in the ostrich.

The aim of this study was to investigate the pharmacokinetics of clenbuterol in the ostrich as no such data is available. Clenbuterol (2 mg) was given as a single oral dose to nine ostriches. Blood samples were collected over a period of 96 h after administration and urine for a period of 5 d. Plasma and urine samples were frozen at -20 degrees C pending analysis. Clenbuterol was quantified using a gas chromatograph-mass selective detector. The method for quantification of clenbuterol in plasma was validated by analysing spiked quality control samples at different concentrations. The limit of quantification was determined to be 0.75 ng ml-1 with an absolute recovery of more than 80%. The geometric mean maximum plasma clenbuterol concentration was 4.40 ng ml-1 with 3.0 h as the median time for maximum concentration. The plasma elimination half-life was 19.7 h. The clenbuterol concentration was above 0.75 ng ml-1 in plasma for 48 h and above 1.0 ng ml-1 in urine for 5 d. These data can be useful in residue analysis for clenbuterol in ostriches.     

Efficiency of four different techniques in coupled HPLC-UV/VIS to quantify overlapping peaks with known spectral features

Summary Four different techniques to quantify unresolved chromatographic peaks with known spectral features combined with photodiode array detection, are investigated as regards their efficiency for the accurate and precise determination of drugs in the low g-range. The comparison includes peak suppression utilising difference chromatograms, first-order derivative chromatograms, selective chromatograms, generated by the calculation of orthogonal polynomial shares, and the powerful least-squares multicomponent analysis approach. Each of these methods uses UV-spectra taken throughout, the peak. The results presented and conclusions reached should enable the chromatographer to come to a decision about the reasonable use of these options now provided by multichannel detection in HPLC.