Benchmarking breath analysis using peppermint approach with gas chromatography ion mobility spectrometer coupled to micro thermal desorber.

The Peppermint Initiative, established within the International Association of Breath Research, introduced the peppermint protocol, a breath analysis benchmarking effort designed to address the lack of inter-comparability of outcomes across different breath sampling techniques and analytical platforms. Benchmarking with gas chromatography-ion mobility spectrometry (GC-IMS) using peppermint has been previously reported however, coupling micro-thermal desorption (µTD) to GC-IMS has not yet, been benchmarked for breath analysis. To benchmarkµTD-GC-IMS for breath analysis using the peppermint protocol. Ten healthy participants (4 males and 6 females, aged 20-73 years), were enrolled to give six breath samples into Nalophan bags via a modified peppermint protocol. Breath sampling after peppermint ingestion occurred over 6 h att= 60, 120, 200, 280, and 360 min. The breath samples (120 cm3) were pre-concentrated in theµTD before being transferred into the GC-IMS for detection. Data was processed using VOCal, including background subtractions, peak volume measurements, and room air assessment. During peppermint washout, eucalyptol showed the highest change in concentration levels, followed byα-pinene andß-pinene. The reproducibility of the technique for breath analysis was demonstrated by constructing logarithmic washout curves, with the average linearity coefficient ofR2= 0.99. The time to baseline (benchmark) value for the eucalyptol washout was 1111 min (95% CI: 529-1693 min), obtained by extrapolating the average logarithmic washout curve. The study demonstrated thatµTD-GC-IMS is reproducible and suitable technique for breath analysis, with benchmark values for eucalyptol comparable to the gold standard GC-MS.

Deep Learning Based Prediction of Gas Chromatographic Retention Indices for a Wide Variety of Polar and Mid-Polar Liquid Stationary Phases.

Prediction of gas chromatographic retention indices based on compound structure is an important task for analytical chemistry. The predicted retention indices can be used as a reference in a mass spectrometry library search despite the fact that their accuracy is worse in comparison with the experimental reference ones. In the last few years, deep learning was applied for this task. The use of deep learning drastically improved the accuracy of retention index prediction for non-polar stationary phases. In this work, we demonstrate for the first time the use of deep learning for retention index prediction on polar (e.g., polyethylene glycol, DB-WAX) and mid-polar (e.g., DB-624, DB-210, DB-1701, OV-17) stationary phases. The achieved accuracy lies in the range of 16-50 in terms of the mean absolute error for several stationary phases and test data sets. We also demonstrate that our approach can be directly applied to the prediction of the second dimension retention times (GC × GC) if a large enough data set is available. The achieved accuracy is considerably better compared with the previous results obtained using linear quantitative structure-retention relationships and ACD ChromGenius software. The source code and pre-trained models are available online.

Current analytical methods for determination of glucosinolates in vegetables and human tissues.

Numerous epidemiological studies have indicated the potential effects of glucosinolates and their metabolites against cancer as well as other non-communicable diseases, such as cardiovascular disease and neurodegenerative disorders. However, information on the presence and quantity of glucosinolates in commonly consumed vegetables and in human fluids is sparse, largely because well-standardised methods for glucosinolate determination are not available, resulting in published data being inconsistent and conflicting. Thus, studies published since 2002 on the most recent developments of glucosinolate extraction and identification have been collected and reviewed with emphasis on determination of the intact glucosinolates by LC-MS and LC-MS/MS. This overview highlights the glucosinolate extraction methods used, the stability of glucosinolates during extraction, the availability of stable isotope labelled internal standards and the use of NMR for purity analysis, as well as the current analytical techniques that have been applied for glucosinolate analysis, e.g. liquid chromatography with mass spectrometric detection (LC-MS). It aims to interpret the findings with a focus on the development of a validated method, which will help to determine the glucosinolate content of vegetative plants and human tissues, and the identification and determination of selected glucosinolate metabolites.

Standardization of chromatographic signals - Part I: Towards obtaining instrument-agnostic fingerprints in gas chromatography.

One of the main causes for the sparse use of multivariate analytical methods in routine laboratory work is the dependency on the measuring instrument from which the analytical signal is acquired. This issue is especially critical in chromatographic equipment and results in limitations of their applicability. The solution to this problem is to obtain a standardized instrument-independent signal -or instrument-agnostic signal- regardless of the measuring instrument or of the state of the same instrument from which it has been acquired. The combined use of both internal and external standard series, allows us to have external and transferable references for the normalization of both the intensity and the position of each element of the data vector being arranged from the raw signal. From this information, a simple mathematical data treatment process is applied and instrument-agnostic signals can be secured. This paper describes and applies the proposed methodology to be followed for obtaining standardized instrumental fingerprints from two significant fractions of virgin olive oil (volatile organic compounds and triacylglycerols), obtained by gas chromatography coupled to mass spectrometry (GC-MS) and analysed with two temperature conditions (conventional and high-temperature, respectively). The results of both case studies show how the instrument-agnostic fingerprints obtained are coincidental, regardless of the state of the chromatographic system or the time of acquisition.

A unique data analysis framework and open source benchmark data set for the analysis of comprehensive two-dimensional gas chromatography software.

Comprehensive two-dimensional gas chromatography (GC × GC) is amongst the most powerful separation technologies currently existing. Since its advent in early 1990, it has become an established method which is readily available. However, one of its most challenging aspects, especially in hyphenation with mass spectrometry is the high amount of chemical information it provides for each measurement. The GC × GC community agrees that there, the highest demand for action is found. In response, the number of software packages allowing for in-depth data processing of GC × GC data has risen over the last couple of years. These packages provide sophisticated tools and algorithms allowing for more streamlined data evaluation. However, these tools/algorithms and their respective specific functionalities differ drastically within the available software packages and might result in various levels of findings if not appropriately implemented by the end users. This study focuses on two main objectives. First, to propose a data analysis framework and second to propose an open-source dataset for benchmarking software options and their specificities. Thus, allowing for an unanimous and comprehensive evaluation of GC × GC software. Thereby, the benchmark data includes a set of standard compound measurements and a set of chocolate aroma profiles. On this foundation, eight readily available GC × GC software packages were anonymously investigated for fundamental and advanced functionalities such as retention and detection device derived parameters, revealing differences in the determination of e.g. retention times and mass spectra.

Identificação de possíveis biomarcadores metabolômicos salivares e vias metabólicas alteradas em pacientes com carcinoma de células escamosas bucal / Identification of possible salivary metabolomic biomarkers and altered metabolic pathways in patients with oral squamous cell carcinoma

O carcinoma de células escamosas bucal (CCEB) representa 90% das neoplasias malignas da região intrabucal. O desenvolvimento dessa doença é multifatorial, envolvendo fatores genéticos e ambientais que podem ser, muitas vezes, desconhecidos e que podem apresentar modificação das vias metabólicas. Assim, o objetivo desta pesquisa foi identificar os metabólitos salivares de pacientes com CCEB na busca de vias metabólicas alteradas, assim como possíveis biomarcadores metabólicos. Os metabólitos salivares de 27 pacientes com CCEB foram comparados aos metabólitos de 41 pacientes controles. As amostras de metaboloma salivar foram analisadas por meio de cromatógrafo gasoso acoplado a um espectrômetro de massa (GC-MS). Os resultados indicaram 26 metabólitos específicos para o grupo CCEB e 5 específicos do grupo controle. No diagrama de Venn foram observados 77 metabólitos comuns aos dois grupos. Esta análise foi crucial para a descoberta de três vias metabólicas com diferença estatística entre os grupos: a lançadeira malato-aspartato, a via do metabolismo da beta-alanina e o efeito Warburg. Todas essas vias relacionam-se com a produção de energia celular na carcinogênese, promovendo um ambiente favorável para o alto consumo energético e sobrevivência celular. Ao comparar os metabólitos salivares de pacientes controles e com câncer bucal, 40 metabólitos foram capazes de distinguir os grupos estatisticamente, sendo 20 metabólitos aumentados nos pacientes com carcinoma e 20 aumentados nos pacientes sem a doença. Com a finalidade de identificar os metabólitos como possíveis biomarcadores salivares, a curva ROC foi traçada e um corte de 0,8 no AUC foi construído descobrindo-se 24 metabólitos. Após um novo corte nos metabólitos com AUC de 0,9, metabólitos como ureia, leucina, inosina e metionina puderam ser explorados como candidatos a biomarcadores metabolômicos salivares de pacientes com CCEB já relatados na literatura. Além destes, 6 outros metabólitos apresentaram AUC acima de 0,9 e são relatados pela primeira vez em um estudo de metaboloma salivar de pacientes sul-americanos com câncer bucal, são estes: o ácido málico, maltose, ácido protocatecuico, lactose, ácido-2-cetoadípico e catecol. Essas alterações metabólicas auxiliam na descoberta de biomarcadores salivares de pacientes com câncer bucal e despertam o interesse para que haja outros estudos com maior número de amostras no intuito de validar nossos resultados(AU)

Oral squamous cell carcinoma (CCEB) represents 90% of intraoral malignant neoplasms. The development of this disease is multifactorial, involving genetic and environmental factors that can often be unknown and can present the modification of metabolic pathways. Thus, the objective of this research was to identify the salivary metabolites of patients with CCEB in the search of altered metabolic pathways, as well as possible metabolic biomarkers. The salivary metabolites of 27 patients with CCEB were compared to the metabolites of 41 control patients. The salivary metabolome samples were analyzed using a gas chromatograph coupled to a mass spectrometer (GC-MS). The results indicated 26 specific metabolites for the CCEB group and 5 specifics for the control group. In the Venn diagram 77 other metabolites common to both groups were observed. This analysis was crucial for the discovery of three metabolic pathways with statistical difference between the groups: the malateaspartate shuttle, the beta-alanine metabolism pathway and the Warburg effect. All of these pathways are related to the production of cellular energy in carcinogenesis, promoting a favorable environment for high energy consumption and cell survival. When comparing the salivary metabolites of control patients with oral cancer, 40 metabolites were able to distinguish the groups statistically, with 20 metabolites increased in patients with carcinoma and 20 increased in patients without the disease. In order to identify the metabolites as possible salivary biomarkers, the ROC curve was drawn and a cohort of 0.8 in the AUC was performed by discovering 24 metabolites. After a new cohort of metabolites with AUC of 0.9, metabolites such as urea, leucine, inosine and methionine could be explored as possible salivary metabolomic biomarkers of patients with CCEBs already reported in the literature. In addition to these, 6 other metabolites showed AUC above 0.9 and are reported for the first time in a study of salivary metabolome in South American patients with oral cancer, these are: malic acid, maltose, protocatechuic acid, lactose, 2-ketoadipic and catechol. These metabolic changes help in the discovery of salivary biomarkers of patients with oral cancer and arouse interest for further studies with a larger number of samples in order to validate our results(AU)

A step forward in the equivalence between thermal and differential-flow modulated comprehensive two-dimensional gas chromatography methods.

Comprehensive two-dimensional gas chromatography (GC×GC) based on flow-modulation (FM) is gaining increasing attention as an alternative to thermal modulation (TM), the recognized GC×GC benchmark, thanks to its lower operational cost and rugged performance. An accessible, rational procedure to perform method translation between the two platforms would be highly valuable to facilitate compatibility and consequently extend the flexibility and applicability of GC×GC. To enable an effective transfer, the methodology needs to ensure preservation of the elution pattern, separation power, and sensitivity. Here, a loop-type thermal modulation system with dual detection (TM-GC×GC-MS/FID) used for the targeted analysis of allergens in fragrances is selected as reference method. Initially, six different columns configurations are systematically evaluated for the flow-modulated counterpart. The set-up providing the most consistent chromatographic separation (20 m x 0.18 mm dc x 0.18 µm df + 1.8 m x 0.18 mm dc x 0.18 µm df) is further evaluated to assess its overall performance in terms of sensitivity, linearity, accuracy, and pattern reliability. The experimental results convincingly show that the method translation procedure is effective and allows successful transfer of the target template metadata. Additionally, the FM-GC×GC-MS/FID system is suitable for challenging applications such as the quantitative profiling of complex fragrance materials.

Selection of calibration compounds for selectivity evaluation of wall-coated, open-tubular columns for gas chromatography by the solvation parameter model.

To facilitate faster selectivity evaluation of wall-coated, open-tubular columns using the solvation parameter model a reduced set of calibration compounds is identified and validated for the temperature ranges 60-140 °C and 160-260 °C. The Kennard-Stone uniform mapping algorithm is used to identify the calibration compounds from a larger database of compounds with known retention properties previously adopted for column selectivity evaluation. Thirty-five compounds for each temperature range are required to minimize the standard deviation of the system constants used for selectivity evaluation and to minimize differences between system constants determined by conventional calibration and the reduced calibration compounds. The models for the reduced calibration compounds on ten siloxane-based and poly(ethylene glycol) stationary phases have a coefficient of determination of 0.984 to 0.998 and standard error of the estimate of 0.012 to 0.30. The predictive capability of models is evaluated for the reduced sets of calibration compounds using external test sets with ranking of the calibration models by changes in the average error, average absolute error and root mean square error of prediction for the test sets. For the selected thirty-five reduced calibration compounds the range for the average absolute error was 0.014 to 0.033 and 0.016 to 0.040 for the root mean square error of prediction for the independent test sets.

A Single-Column Gas Chromatography Method for Quantifying Toxic Alcohols.

BACKGROUND: Rapid identification and quantification of toxic alcohols and ethylene glycol is imperative for appropriate treatment. Clinical laboratories frequently rely on direct injection gas chromatography (GC) methods, but these methods require inlet maintenance and multiple GC systems. To overcome these challenges, we developed a single-column headspace GC method for both toxic alcohols and glycols that streamlines patient sample analysis for toxic alcohol ingestion. METHODS: Optimal parameters for nonderivatized (volatile) and derivatized (glycol) plasma samples were determined using a 7890 A headspace sampler, an Agilent 7697 A GC system, a DB-200 column, and a flame ionization detector. Limit of Quantification (LoQ), linearity, imprecision, carry-over, method comparison, and interference studies were performed using quality control materials and prepared plasma samples. RESULTS: Our volatile method is linear to 3000 mg/L (ethanol) with LoQ concentrations below 20 mg/L (ethanol). The glycol method is linear to 2000 mg/L (ethylene glycol) with LoQ concentrations below 40 mg/L (ethylene glycol). Total assay impression ranged from 1.7% for ethanol to 13.3% for propylene glycol. Both methods were free of sample carryover and compared favorably with a similar clinical method at an outside laboratory. Propionic acid, an accumulating metabolite in methylmalonic acidemia that interferes with ethylene glycol identification by a different method, did not interfere with the ethylene glycol method reported here. CONCLUSIONS: Our single-column headspace GC method provides reliable, robust, and rapid identification and quantification of commonly encountered toxic alcohols. Clinical laboratories relying on direct injection Gas Chromatography (GC) for toxic alcohol analysis face challenges including frequent inlet maintenance, sample carryover, or the need for separate GC systems for volatile and glycol analysis. We summarize our development and optimization of two headspace GC methods for nonderivatized (volatile) and derivatized (glycol) plasma samples that use a single DB-200 analytical column. These methods are comparable to other GC methods, not prone to sample carryover, eliminate the need for multiple GC systems or columns, and are readily applicable to other laboratories that provide toxic alcohol analysis.

Homogeneity assessment of reference materials for sensory analysis of liquid foodstuffs. The virgin olive oil as case study.

Sensory properties are critical characteristics that determine quality and can be evaluated by trained tasting panels. The panels function as multi-sensor measuring instrument and need the use of reference materials (RMs) for training. The homogeneity between units packaged from a batch of RM can be evaluated by gas chromatography coupled to flame ionization detection (GC-FID), using this instrumental technique as an alternative to sensory analysis. For this purpose, the fingerprint methodology is applied, taking into account that the homogeneity assessment will be based on evaluating the similarity between the fingerprints of the fraction of volatile organic compounds acquired from samples representative of the batch. The proposed methodology is applied with good results to evaluate the homogeneity of several RMs for sensory analysis of virgin olive oil samples, using similarity indices, control charts and exploratory analysis of multivariate data to observe the grouping RM and fingerprint regions representative of each defect.

False-Positive Enzymatic Alcohol Results in Perimortem Specimens.

Herein, we present 2 cases referred to the North Carolina Office of the Chief Medical Examiner (NC OCME) in which ethanol results reported by different hospital laboratories, using alcohol dehydrogenase (ADH)-based assays, were positive, whereas results of headspace gas chromatography testing performed in the NC OCME laboratory were negative. Literature reports suggest that false-positive ethanol measurements from ADH-based assays can occur when a combination of elevated lactate and lactate dehydrogenase (LD) are present in the specimen. The results were reported in perimortem specimens collected from 2 children with unrelated medical conditions. The cases and associated clinical parameters are considered based on the lactate/LD explanation for the false-positive results, to facilitate the recognition of circumstances that can produce erroneous serum ethanol results.

Reference Intervals of Serum Non-Cholesterol Sterols by Gender in Healthy Japanese Individuals.

AIMS: The present study was conducted to establish a practical method for measuring non-cholesterol sterols and reference intervals of serum levels. METHODS: Healthy subjects (109 men and 151 women), four patients with sitosterolemia, and 10 heterozygous mutation carriers of ABCG5/ABCG8 genes were investigated. Then, three non-cholesterol sterols (sitosterol, campesterol, and lathosterol) of fasting serum samples were measured via a practical and highly sensitive gas chromatography (GC) method with 0.2 µg/mL as the lower limit of quantification. The coefficient of variation (CV) values for within-run reproducibility were 3.06%, 1.89%, and 1.77% for lathosterol, campesterol, and sitosterol, respectively. The CV values for between-run reproducibility were 2.81%, 2.06%, and 2.10% for lathosterol, campesterol, and sitosterol, respectively. RESULTS: The serum levels of sitosterol and campesterol were significantly higher in women than in men, whereas the serum levels of lathosterol were significantly higher in men than in women. Because of these gender difference, the determination of reference intervals of the three sterol values was performed by considering gender. The reference intervals of sitosterol, campesterol, and lathosterol were 0.99-3.88, 2.14-7.43, and 0.77-3.60 µg/mL in men and 1.03-4.45, 2.19-8.34, and 0.64-2.78 µg/mL in women, respectively. The serum levels of sitosterol and campesterol were higher in patients with sitosterolemia (94.3±47.3 and 66.3±36.6 µg/mL, respectively) than in healthy subjects. CONCLUSION: These results demonstrate a practical and highly sensitive GC method to measure non-cholesterol sterol levels and gender-segregated reference intervals of sitosterol, campesterol, and lathosterol in Japanese healthy subjects.

Identifying and Mitigating Errors in Screening for Organic Extractables and Leachables: Part 1-Introduction to Errors in Chromatographic Screening for Organic Extractables and Leachables and Discussion of the Errors of Omission.

Substances leached from materials used in pharmaceutical manufacturing systems, packages, and/or medical devices can be administered to a patient as part of a clinical therapy. These leachables can have an undesirable effect on the effectiveness of the therapy and/or patient safety. Thus, relevant samples such as material extracts or drug products are chromatographically screened for foreign organic impurities, where screening is the analytical process of discovering, identifying, and quantifying these unspecified foreign impurities. Although screening methods for organic extractables and leachables have achieved a high degree of technical and practical sophistication, they are not without issues with respect to their ability to accomplish the aforementioned three functions. In this first part of a series of three manuscripts, the process of screening is examined, limitations in screening are identified, and the concept of using an internally developed analytical database to identify, mitigate, or correct these errors is introduced. Furthermore, errors of omission are described, where an error of omission occurs when a screening method fails to produce a recognizable response to an analyte present in the test sample. The error may be that no response is produced ("falling through the cracks") or that a produced response is not recognizable ("failing to see the tree for the forest"). In either case, proper use of a robust internal extractables/leachables database can decrease the frequency with which errors of omission occur. Examples of omission errors, their causes, and their possible resolution are discussed.

Problem in the molecular area of polar probe molecules used in inverse gas chromatography.

Although the inverse gas chromatography technique has been used to measure the surface acid-base properties for almost 50 years, several basic problems still hinder its further development and application. One of the problems is that for the polar probe molecules of dichloromethane, acetone, ethyl acetate, tetrahydrofuran, and ethanol, the molecular area of each probe has different values in literature. In this paper, we point out the incorrect molecular area values presented in the literature by comparing these values with those indicated in Smallwood's Handbook of Organic Solvent Properties. The correct molecular area values of 11 polar probes are determined by plotting the molecular areas of the polar probes versus the van der Waals surface areas reported in the handbook. The correct molecular areas for six common polar probes, namely, dichloromethane, trichloromethane, acetone, ethyl acetate, diethyl ether, and tetrahydrofuran, are 38.0, 44.0, 42.5, 48.0, 47.0, and 45.0 Å2, respectively. Benzene is a specific molecule with a conjugated π-bond structure, and its molecular area is estimated to be larger than that of other molecules. Therefore, benzene is unsuitable for use as a probe molecule.

Gas chromatography system constant database over an extended temperature range for nine open-tubular columns.

The solvation parameter model is used to characterize the retention properties of poly(octylmethylsiloxane), poly(dimethyldiphenylsiloxane), silphenylene-dimethylsiloxane copolymer, poly(dimethylmethyltrifluoropropylsiloxane), poly(3-cyanopropylphenyldimethylsiloxane), and poly(ethylene glycol) (SPB-Octyl, HP-5, Rxi-17, Rxi-5Sil MS, Rtx-OPP, DB-1701, DB-225, Rtx-440 and HP-INNOWAX) stationary phases over an extended temperature range from 60 °C to close to the maximum column operating temperature. System maps constructed from the system constant database are used to evaluate the influence of temperature on the contribution of intermolecular interactions to differences in selectivity. None of the stationary phases are hydrogen-bond acids. Increasing temperature reduces the contribution from cavity formation/dispersion, dipole-type interactions and hydrogen-bond basicity in a stationary phase specific manner with their preservation to the highest temperature studied for each column. Electron lone pair interactions are generally repulsive at lower temperatures becoming attractive and leading to an increase in retention at higher temperatures.

A strategy for validating concentrations of oxylipin standards for external calibration.

Quantitative analysis of oxylipins by means of chromatography/mass spectrometry is based on (external) calibration with standard compounds. Therefore, the quality of analytical standards is of fundamental importance for accurate results. Recently launched certified standards with an assured concentration within a narrow range are useful tools to verify analytical standards. However, such standards are only available for a few compounds. Based on the exemplary comparison of certified with none certified standards we suggest a tiered approach to validate and control the concentrations when preparing an external calibration based on non-certified oxylipin standards. Concentrations are evaluated by means of liquid chromatography negative electrospray ionization mass spectrometry (LC-ESI(-)-MS) in selected ion monitoring mode and UV spectroscopy. Based on the suggested approach, more than 50% of the standards in our calibration mix could be validated. Though most of the non-certified standards are of good quality, several oxylipin concentrations differ considerably demonstrating that a quality control strategy as suggested here is a mandatory prerequisite for quantitative oxylipin metabolomics.

Development and Validation of an Analytical Method to Quantitate Tris(chloroisopropyl)phosphate in Rat and Mouse Plasma using Gas Chromatography with Flame Photometric Detection.

Tris(chloropropyl)phosphate (TCPP) is an organophosphorus flame retardant (OPFR) and plasticizer increasingly used in consumer products and as a replacement for brominated flame retardants. Commercially available TCPP is a mixture of four structural isomers the most abundant of which is tris(1-chloro-2-propyl)phosphate (TCPP-1). Although there is a widespread use of TCPP and potential for human exposure, there is limited data on the safety or toxicity of TCPP. The National Toxicology Program is conducting long-term studies to examine the toxicity of the TCPP in rats after lifetime exposure, including perinatal oral exposure. Quantitative estimates of internal dose are essential to interpret toxicological findings in rodents. To aid in this, a method was fully validated to quantitate the most abundant isomer, TCPP-1, in female Harlan Sprague Dawley (HSD) rat and B6C3F1 mouse plasma with partial validation in male rat plasma, and male and female mouse plasma. The method used protein precipitation using trichloroacetic acid followed by the extraction with toluene, and analysis by gas chromatography with flame photometric detection. The performance of the method was evaluated over 5-70 ng TCPP-1/mL plasma. The method was linear (r ≥ 0.99), accurate (inter-day relative error: ≤ ± -7.2) and precise (inter-batch relative standard deviation: ≤27.5%). The validated method has lower limits of quantitation and detection of ~5 and 0.9 ng/mL, respectively, in female HSD rat plasma and can be used on samples as small as 50 µL demonstrating the applicability to plasma samples from toxicology studies.

Development and validation of analytical methodology by GC-FID using hexadecyl propanoate as an internal standard to determine the bovine tallow methyl esters content.

EN 14103:2003 and EN 14103:2011 were developed in order to determine fatty acid methyl ester (FAME) content of biodiesel. The internal standards (IS) of biodiesel include methyl heptadecanoate (MHD) and methyl nonadecanoate (MND), respectively. However, since these ISs are also present in bovine tallow methyl esters (BTME) or overlapping peaks, they have not been efficient. This work proposes an improved BTME determination method by using hexadecyl propanoate (HDP) as an IS. For this purpose, an analytical methodology by Gas Chromatography-Flame Ionization Detector (GC-FID) was developed and validated, where HDP demonstrated selectivity in retention time between peaks C16:1 and C18:0 for coconut and soybeans methyl esters and BTME, as well as resolution >1.5 for the BTME in split mode 30:1. Trueness in the determination of BTME content using the HDP as an IS was statistically equivalent to confidence interval of 95% for the null hypothesis statistic test, even when only 20% of the HDP was utilized in comparison with the IS concentrations defined by EN 14103:2003 and EN 14103:2011. This allowed the biodiesel analysis to be performed five times more with 1 g of HDP. Furthermore, the method developed enabled us to reduce the analysis time by 21.6%, without prejudice to the integration of peaks (C6:0 to C24:1). Regarding the repeatability and intermediate precision tests, results of RSD (%) ≤ 2% were reached. Additionally, the method developed has proved to be robust. HDP is a long-chain fatty alcohol ester absent from feedstocks used in biodiesel synthesis. It presents all of the characteristics for a good IS, ideal for application via internal standardization method, as recommended by EN 14103.

Variability on ethyl glucuronide concentrations in hair depending on sample pretreatment, using a new developed GC-MS/MS method.

Quantitative determination of ethyl glucuronide in keratin matrix, particularly in hair samples, provides a significant contribution to the evaluation of the extent of ethanol intake. The first-choice method to carry out this analysis is LC-MS/MS, but other techniques may be used. The aim of this work is: a) to develop and validate a GC-MS/MS method for ethyl glucuronide determination in hair; b) to compare GC-MS/MS and LC-MS/MS in analysis of real samples; c) to compare EtG concentration obtained after hair cutting and pulverization. About 30 mg hair samples were washed, pulverized and soaked in 1 ml deionized water. After incubation, the solution was purified through a SPE anion exchange cartridge; the eluate was dried under nitrogen stream, derivatized with PFPA and reconstituted in n-hexane. Then, the sample was injected in the GC-MS/MS system, operating in negative chemical ionization mode and in selected reaction monitoring. The two most intense transitions were used to monitor ethyl glucuronide and deuterated internal standard. All the validation parameters fulfilled the international acceptance criteria. LOD and LOQ were set at 2.0 and 3.0 pg/mg respectively. This method was applied to 194 hair samples collected from teetotallers and alcohol consumers and represents a suitable alternative to LC-MS/MS for the determination of EtG in hair samples, in particular when scarce quantity of hair is available. This study confirmed that pulverization of hair increases the concentration of EtG, but some variability of EtG levels remains probably due to the presence of non-homogeneous material even though pulverization.

Polar and non-polar intracellular compounds from microalgae: Methods of simultaneous extraction, gas chromatography determination and comparative analysis.

A method to simultaneously extract polar (PC) and non-polar compounds (NPC) from microalgae was developed for further determination of intracellular metabolites by gas chromatography. The proposed method was validated and used to characterize two Chlorophyceae, Chlorella vulgaris and Scenedesmus obliquus, and two Cyanobacteria, Aphanothece microscopica Nagëli and Phormidium autumnale. The compounds were extracted with a reduced amount of organic solvent mixture (methanol-chloroform), compared to the reference method, under different conditions of homogenization and/or cell disruption. The NPC were derivatized by acid catalysis, whereas the PC fraction was derivatized using N-methyl,N-tert-Butyldimethylsilyltrifluoroacetamide (MTBSTFA) in alkaline medium. The following parameters for method validation were considered: selectivity, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, and accuracy. All methods of homogenization and cell disruption extracted both PC and NPC from Chlorophyceae and Cyanobacteria. Derivatization of PC presented satisfactory validation parameters. Eleven fatty acids, six free amino acids, and three organic acids were found within the evaluated microalgae species, succinic, malic, and citric acids, important intermediates of the tricarboxylic acid cycle. Glutamic acid was the amino acid found in greatest quantities in all species. Chlorophyceae presented a higher concentration of unsaturated fatty acids, while Cyanobacteria had more saturated fatty acids. Thus, the proposed method was suitable to metabolically characterize both PC and NPC from microalgae.