Rapid and reliable quantification of urinary malondialdehyde by HILIC-MS/MS: A derivatization-free breakthrough approach.

BACKGROUND: The development of fast analytical methods is crucial for the research, discovery, and confirmation of crucial biomarkers. Furthermore, the implementation of fast analytical strategies contributes to efficient and time-effective procedures. In this sense, analysis of malondialdehyde (MDA) has become an important tool for understanding the role of oxidative stress in various diseases and for evaluating the efficacy of therapeutic interventions. RESULTS: A rapid and robust liquid chromatography tandem mass spectrometry method (HPLC-MS/MS) has been developed to determine endogenous amounts of malondialdehyde (MDA) in human urine without any associated derivatization reaction. MDA was separated in 4 min through a Urea-HILIC column and was analyzed using a triple quadrupole mass spectrometer in negative electrospray ionization mode. With a 50-fold dilution as the only sample pretreatment after alkaline hydrolysis, no matrix effect was present, which allowed for a fast and simple quantification by means of an external standard calibration with a limit of detection of 0.20 ng mL-1. The whole methodology was validated by analyzing unspiked and spiked urine samples from ten healthy individuals and comparing with the results obtained by the standard addition method. MDA was detected in all cases, with natural concentrations varying from 0.11 ± 0.03 to 0.31 ± 0.03 mg g-1 creatinine. Accuracies were found to be satisfactory, ranging from 95 % to 101 %. The proposed method also exhibited good repeatability and reproducibility (RSD<15 %) for four quality control levels. SIGNIFICANCE: The main significance of this method is the avoidance of a derivatization reaction for the determination of urinary MDA, this constituting a step forward when compared with previous literature. This breakthrough not only streamlines time analysis to less than 5 min per sample but also results in a more robust procedure. Consequently, the method here developed could be applied to subsequent future research involving the determination of MDA as a lipid peroxidation biomarker, where simple, rapid, and reliable methods could represent a significant improvement.

Multiple headspace sampling coupled to a programmed temperature vaporizer to improve sensitivity in headspace-gas chromatography. Determination of aldehydes.

The improvement of sensitivity in headspace (HS) sampling of not very volatile analytes constitutes a challenge that has usually been approached through coupling with additional techniques. Here we propose a new methodology for increasing sensitivity through a multistep approach. This proof of concept is based on direct coupling of a headspace sampler with a programmed temperature vaporizer (PTV) and a gas chromatograph (GC), with mass spectrometry (MS) detection. Analytes are extracted from the same vial in a stepwise procedure, splitting the headspace generation time of conventional HS into four periods and using the PTV to cryogenically trap the analytes during the successive HS samplings. Solvent vent mode is mandatory in order to retain the analytes, purging the gas solvent at an adequate initial low temperature and flash-heating the PTV liner in a quick ramp (720 °C/min), once the HS samplings are finished. Linear aldehydes, from pentanal to decanal, possible biomarkers of several diseases have been selected as model compounds. This multiple HS method has been compared with conventional HS, and it has been validated in terms of linearity, limits of detection, repeatability, reproducibility and accuracy. The limits of detection (LOD) ranged from 0.004 to 0.159 µg/L. Enrichment factors (EF) in relation to the conventional HS method ranged from 3.0 to 6.7, except for pentanal (EF: 0.8), which is too volatile and polar to be trapped in the PTV with the multiple HS methodology. Similar enrichment factors were obtained in a urine sample.

Derivatization coupled to headspace programmed-temperature vaporizer gas chromatography with mass spectrometry for the determination of amino acids: Application to urine samples.

A new method based on headspace programmed-temperature vaporizer gas chromatography with mass spectrometry has been developed and validated for the determination of amino acids (alanine, sarcosine, ethylglycine, valine, leucine, and proline) in human urine samples. Derivatization with ethyl chloroformate was employed successfully to determine the amino acids. The derivatization reaction conditions as well as the variables of the headspace sampling were optimized. The existence of a matrix effect was checked and the analytical characteristics of the method were determined. The limits of detection were 0.15-2.89 mg/L, and the limits of quantification were 0.46-8.67 mg/L. The instrumental repeatability was 1.6-11.5%. The quantification of the amino acids in six urine samples from healthy subjects was performed with the method developed with the one-point standard additions protocol, with norleucine as the internal standard.

In situ derivatization combined to automated microextraction by packed sorbents for the determination of chlorophenols in soil samples by gas chromatography mass spectrometry.

A method based on the coupling of in situ extraction and derivatization of chlorophenols (CPs) (2-chlorophenol, 4-chloro-3-methylphenol, 2,4-dichlorophenol, and 2,4,6-trichlorophenol) from soils, accomplishing their preconcentration by means of automated microextraction by packed sorbent (MEPS), is proposed. After extraction and acylation of the chlorophenols in aqueous medium, the liquid phase obtained is subjected to the MEPS procedure. The QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) and MEPS techniques were compared and the results confirmed the preconcentration carried out with MEPS. The existence of a matrix effect was checked and the analytical characteristics of the method were determined in a soil sample. The method provided good linearity (from 1 to 12µgkg(-1)), together with good repeatability and reproducibility values (RSD equal to or less than 10%). The limits of detection were in the 0.118-0.894µgkg(-1) range. A certified reference material was applied to validate the proposed methodology.

Ionic liquids as stationary phases in gas chromatography: determination of chlorobenzenes in soils.

The present research focuses on the evaluation of different ionic liquid (IL) stationary phases in gas chromatography. The different IL columns were evaluated in terms of peak resolution (Rs) and peak symmetry for the separation of the chlorobenzenes. The determination of chlorobenzenes in soil samples by means of the optimal IL stationary phase (SLB-IL82) is proposed as an application. Soil pretreatment was based on a simplified quick, easy, cheap, effective, rugged, and safe extraction procedure and a large injection volume via a programed temperature vaporizer working in solvent vent mode. The retention time of the chlorobenzenes increased as the polarity of the IL column decreased. SLB-IL82 is the stationary phase that provides the best values as regards Rs and asymmetry factor. Soil sample blanks were spiked with the analytes before subjecting the sample to the extraction process. The existence of a matrix effect was checked and the analytical characteristics of the method were determined in a fortified garden soil sample. The method provided good linearity, good repeatability and reproducibility values, and the LODs were in the 0.1-4.7 µg/kg range. Two fortified soil samples were applied to validate the proposed methodology.

Coupling of microextraction by packed sorbents with gas chromatography with ionic liquid stationary phases for the determination of haloanisoles in wines.

A new method based on the coupling of microextraction by packed sorbents (MEPS) and gas chromatography with ionic liquid stationary phases for the determination of four haloanisoles in wine matrices is proposed. The analytes were concentrated in a C18 barrel insert and needle (BIN), desorbed with a small volume of an organic solvent, and determined by gas chromatography with electron-capture detection (GC-µECD). The influence of several factors on the efficiency of extraction, washing and elution steps was investigated. Additionally, ionic liquid stationary phases of very different polarities were evaluated in terms of efficiency. Under optimized conditions, the analytes were first extracted in the sorbent material and, after an adequate washing step, eluted with 50 µL of ethanol. An aliquot of 5 µL of this extract was further injected, using a programmed temperature vaporizer (PTV), into the chromatographic system. The yield of the overall procedure ranged from 48 to 60%; the reproducibility of the method, calculated as the relative standard deviation (RSD), was below 10% for all compounds, and the limits of detection ranged from 1.2 to 4.8 ng L(-1). External calibration was used in the determination of the target compounds in wine samples.

A simplified Quick, Easy, Cheap, Effective, Rugged and Safe approach for the determination of trihalomethanes and benzene, toluene, ethylbenzene and xylenes in soil matrices by fast gas chromatography with mass spectrometry detection.

A method based on simplified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction followed by large-injection volume-fast gas chromatography and mass spectrometry detection has been developed for the determination of trihalomethanes (chloroform, bromodichloromethane, dibromochloromethane and bromoform) and BTEX (benzene, toluene, ethylbenzene and xylenes) in soil samples. The simplified version of QuEChERS used meets the requirements of the "green chemistry" and provides reliable results with high sample throughput, low solvent consumption, little labour and the use of materials commonly employed in laboratories. The GC device used is equipped with a programmable temperature vaporizer (PTV), with a liner packed with Tenax-TA(®). Using the solvent-vent mode, the PTV allows the injection of large volumes of sample, affording an improvement in the sensitivity of the method. The chromatographic conditions used here allowed the separation of the compounds in less than 5.50 min. Good linearity was obtained for all the target compounds, with highly satisfactory repeatability and reproducibility values. The limits of detection were in the 0.2 to 15 µg kg(-1) range. The method was validated by the analysis of two certified reference materials.

Determination of antioxidants in new and used lubricant oils by headspace-programmed temperature vaporization-gas chromatography-mass spectrometry.

A sensitive method is presented to determine antioxidants (2-, 3-, and 4-tert-butylphenol, 2,6-di-tert-butylphenol, 3-tert-butyl-4-hydroxyanisol, 2,6-di-tert-butyl-4-methylphenol, 1-naphthol, and diphenylamine) in new and used lubricant oil samples. Research was carried out on a GC device equipped with a headspace sampler, a programmed temperature vaporizer, and an MS detector unit. The proposed method does not require sample treatment prior to analyses, hence eliminating possible errors occurring in this step. Sample preparation is reduced when placing the oil sample (2.0 g) in the vial and adding propyl acetate (20 µL). Solvent vent injection mode permits a pre-concentration of the compounds of interest in the liner filled with Tenax-TA®, while venting other species present in the headspace. Thereby, both the life of the liner and the capillary column is prolonged, and unnecessary contamination of the detector unit is avoided. Calibration was performed by adding different concentrations of analytes to a new oil which did not contain any of the studied compounds. Limits of detection as low as 0.57 µg/L (2-tert-butylphenol) with a precision lower or equal to 5.3% were achieved. Prediction of the antioxidants in new oil samples of different viscosities (5W40, 10W40, and 15W40) was accomplished with the previous calibration, and the results were highly satisfactory. To determine antioxidants in used engine oils, standard addition method was used due to the matrix effect.

Determination of trihalomethanes in soil matrices by simplified quick, easy, cheap, effective, rugged and safe extraction and fast gas chromatography with electron capture detection.

A method based on QuEChERS extraction is proposed for the determination of trihalomethanes (chloroform, bromodichloromethane, dibromocloromethane and bromoform) in soil samples. The new version of QuEChERS adapted to soil samples consists of liquid extraction with ethyl acetate, the addition of water to moisten the samples, salting-out partitioning of the water with anhydrous MgSO4, and direct injection of the organic extract, obtained after the centrifugation step, into the gas chromatograph. This simplified extraction procedure maintains the advantages of the original method and avoids some steps, making the final procedure simpler, faster, and cheaper, with the consequent reduction in errors associated with sample manipulation. The experimental conditions of the analytical method, based on fast gas chromatography (FGC) and micro-electron capture detection (microECD), were optimized. The column and oven program used allowed fast separation of the compounds in less than 4 min and the total analysis cycle time was as short as 10 min. The existence of a matrix effect was checked and the analytical conditions of the method were studied in a fortified garden soil sample. The highly sensitive and selective detector used afforded to detection limits in the order of ng/kg for the target compounds. To validate the proposed method two certified reference materials (CRMs) were analyzed.

Determination of trihalomethanes in water samples: a review.

This article reviews the most recent literature addressing the analytical methods applied for trihalomethanes (THMs) determination in water samples. This analysis is usually performed with gas chromatography (GC) combined with a preconcentration step. The detectors most widely used in this type of analyses are mass spectrometers (MS) and electron capture detectors (ECD). Here, we review the analytical characteristics, the time required for analysis, and the simplicity of the optimised methods. The main difference between these methods lies in the sample pretreatment step; therefore, special emphasis is placed on this aspect. The techniques covered are direct aqueous injection (DAI), liquid-liquid extraction (LLE), headspace (HS), and membrane-based techniques. We also review the main chromatographic columns employed and consider novel aspects of chromatographic analysis, such as the use of fast gas chromatography (FGC). Concerning the detection step, besides the common techniques, the use of uncommon detectors such as fluorescence detector, pulsed discharge photoionization detector (PDPID), dry electrolytic conductivity detector (DELCD), atomic emission detector (AED) and inductively coupled plasma-mass spectrometry (ICP-MS) for this type of analysis is described.

Headspace-programmed temperature vaporizer-fast gas chromatography-mass spectrometry coupling for the determination of trihalomethanes in water.

A new method based on the use of a headspace autosampler in combination with a GC equipped with a programmable temperature vaporizer (PTV) and an MS detector has been developed for the screening and quantitative determination of trihalomethanes (THMs) in different aqueous matrices. The use of headspace generation to introduce the sample has the advantage that no prior sample treatment is required, thus minimizing the creation of analytical artifacts and the errors associated with this step of the analytical process. The PTV inlet used was packed with Tenax-TA. The injection mode was solvent vent, in which the analytes are retained in the hydrophobic insert packing by cold trapping, while the water vapour is eliminated through the split line. This allows rapid injection of the sample in splitless mode, very low detection limits being achieved without the critical problem of initial sample bandwidth. The capillary column used allowed rapid separations with half-height widths ranging from 1.68 s (chloroform) to 0.66 s (bromoform). The GC run time was 7.3 min. The use of mass spectrometry allows the identification and quantification of the analytes at the low ppt level. The S/N ratio was at least 10-fold higher when the SIM mode was used in data acquisition as compared to the scan mode. The proposed method is extremely sensitive, with detection limits ranging from 0.4 to 2.6 ppt.

Analysis of class 1 residual solvents in pharmaceuticals using headspace-programmed temperature vaporization-fast gas chromatography-mass spectrometry.

A sensitive method is presented for the fast screening and determination of residual class 1 solvents (1,1-dichloroethene, 1,2-dichloroethane, 1,1,1-trichloroethane, carbon tetrachloride and benzene) in pharmaceutical products. The applicability of a headspace (HS) autosampler in combination with GC equipped with a programmed temperature vaporizer (PTV) and a MS detector is explored. Different injection techniques were compared. The benefits of using solvent vent injection instead of split or splitless-hot injection for the measurement of volatile compounds are shown: better peak shapes, better signal-to-noise ratios, and hence better detection limits. The proposed method is extremely sensitive. The limits of detection ranged from 4.9 ppt (benzene) to 7.9 ppt (1,2-dichloroethane) and precision (measured as the relative standard deviation) was equal to or lower than 12% in all cases. The method was applied to the determination of residual solvents in nine different pharmaceutical products. The analytical performance of the method shows that it is appropriate for the determination of residual class 1 solvents and has much lower detection limits than the concentration limits proposed by the International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use. The proposed method achieves a clear improvement in sensitivity with respect to conventional headspace methods due to the use of the PTV.

Use of mass spectrometry methods as a strategy for detection and determination of residual solvents in pharmaceutical products.

In the present work a strategy for the qualitative and quantitative analysis of residual solvents in pharmaceutical products is reported. First, a low-resolution chromatogram is generated for the identification of the solvents present in the samples by means of headspace generation-fast gas chromatography-mass spectrometry (HS-fast GC/MS). From the plotting of this information by means of contour plots with time and mass/charge axes, it is decided whether quantification of such compounds can be accomplished without chromatographic separation or whether it should be done by fast gas chromatography. The nonseparative method is based on direct coupling of a headspace sampler with a mass spectrometer (HS-MS) and requires a signal recording time of only 3 min, while with fast gas chromatography the time required to obtain a chromatogram is 7.16 min. The use of headspace generation for introducing the sample and standard addition as a quantification technique provided satisfactory results and minimized the matrix effect. An important advantage of the methodologies used here is related to the fact that no prior treatment of the sample is required, thus minimizing the creation of analytical artifacts and the errors associated with this step of the analytical process. The methods were applied to the determination of residual solvents in 27 different pharmaceutical products. Detection and quantitation limits were sufficiently low to enable the estimation of organic volatile impurities according to the International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use.

Determination of benzene in gasoline using direct injection-mass spectrometry.

A high-speed determination of benzene in gasoline samples using a non-separative method based on direct injection into the mass spectrometer is proposed. The results obtained are very similar to those provided with fast GC-MS. The calibration set was made up of gasoline samples in which the benzene was determined chromatographically and samples of gasoline subjected to a process of evaporation--until the complete disappearance of the original benzene--to which known concentrations of this compound had been added. A PLS1 multivariate calibration model was constructed. Cross-validation was used to select the optimum number of PLS components. The prediction capacity of the model was checked with an additional group of gasoline samples that had not been used either in the construction or in the validation of the model. With the direct injection method proposed here it was possible to analyse 24 samples over a period of 1h. The direct injection method is rapid, simple and--in view of the results--highly suitable for the determination of benzene in gasoline samples.

Factors affecting signal intensity in headspace mass spectrometry for the determination of hydrocarbon pollution in beach sands.

One of the main limitations to the use of direct coupling of headspace mass spectrometry (HS-MS) for the quantitative determination of analytes in a sample is related to factors affecting the signal intensity. The importance of strategies aimed at compensating this problem is considerable in the case of classification, and is indeed critical as regards the problems involved in quantification. This paper reports the effects of the different factors affecting HS-MS signal intensity in the quantification of the pollution of beach sands by hydrocarbons--the matrix effect, signal instability over time and nature of the different pollutants present in the polluted sands--and proposes possible solutions. Signal instability was solved by using a multiplicative calibration transfer algorithm. A three-factor Box-Behnken experimental design was used to study the matrix effect, mainly as regards the moisture of the samples, and the results are discussed.

Detection of soil pollution by hydrocarbons using headspace-mass spectrometry and identification of compounds by headspace-fast gas chromatography-mass spectrometry.

The direct coupling of a headspace sampler with a mass spectrometer is proposed as a screening tool for the rapid detection of soil pollution by hydrocarbons from petroleum and derivatives. The samples are subjected to the headspace generation process, with no prior treatment, and the volatiles generated are introduced directly into the mass spectrometer, thereby obtaining a fingerprint of the sample analysed. Suitable treatment of the signal by chemometric techniques allows unequivocal characterisation of the different types of sample. The use of fast gas chromatography with a mass spectrometer detector coupled to the headspace sampler allows identification of the major hydrocarbons present in the mineral and organic polluted samples, interpretation of the results obtained, and demonstrates the analytical potential of headspace-mass spectrometry coupling.

Determination of methyl tert-butyl ether in gasoline: a comparison of three fast methods based on mass spectrometry.

A high-speed quantitative analysis of methyl tert-butyl ether (MTBE) using three different methods with mass spectrometry detection has been performed. The first method is based on fast chromatography and required an analysis time of 5.23 min per sample, although a certain period (6 min) was necessary for the initial measurement conditions to be regained prior to analysing the next sample. The other two are non-separative methods and are based on direct injection and headspace generation. The analysis times were 1.5 and 3.5 min, respectively, although in the latter case an additional period of time was required to extract volatiles from the sample. The analytical characteristics of all three methods are highly satisfactory in terms of linearity, lack of fit, precision and accuracy. The methods were applied to the determination of MTBE in different gasoline samples. The non-separative methods afforded slightly higher concentrations than those found when fast chromatography was used; this is due to the presence of other minor components that contribute to the abundance of the ion at m/z 73, characteristic of MTBE. We propose a correction that removes this error very satisfactorily and allows the same results to be obtained with all three methodologies proposed.

Calibration transfer for solving the signal instability in quantitative headspace-mass spectrometry.

It is reported that calibration transfer is able to compensate the variations in sensitivity in direct coupling of a headspace sampler to a mass spectrometer when used for quantification purposes using multivariate calibration techniques. This strategy of signal stability compensation allows the use of models constructed from large calibration standard sets without having to repeat their measurement even though variations occur in sensitivity, which may or may not be constant along the mass range. This technique offers advantages over the use of internal standards in this methodology and only requires the measurement of a small number of transfer samples with each set of unknown samples. The results obtained in the determination of six volatile organic compounds-benzene, toluene, ethylbenzene, and m-xylene (BTEX), methyl tert-butyl ether (MTBE), and mesitylene-are reported. To obtain an appropriate calibration set, a Plackett-Burman design with five levels of concentration for each component was employed. A PLS multivariate calibration model was constructed with a group of 25 samples. For selection of the optimum number of principal components, an external validation set (5 samples) was used and the prediction capacity of this set was checked with an additional group of samples that had not been used either in the construction or in the validation of the model. The results obtained can be considered highly satisfactory, and the methodology was successfully tested with natural matrixes (river and tap water).

Detection of adulterants in olive oil by headspace-mass spectrometry.

In the present work, we propose the use of direct coupling of a headspace sampler to a mass spectrometer for the detection of adulterants in olive oil. Samples of olive oils were mixed with different proportions of sunflower oil and olive-pomace oil, respectively, and patterns of the volatile compounds in the original and mixed samples were generated. Application of the linear discriminant analysis technique to the data from the signals was sufficient to differentiate the adulterated from the non-adulterated oils and to discriminate the type of adulteration. The results obtained revealed 100% success in classification and close to 100% in prediction. The main advantages of the proposed methodology are the speed of analysis (since no prior sample preparation steps are required), low cost, and the simplicity of the measuring process.