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Inside the world of chemometrics, a fundamental role is played by the experimental design. Although introduced almost a century ago (1935), it is still not widely employed by chemists and its usefulness continues to be underestimated. When asking why, the answers are often the following: it is too difficult to apply and too much experimental effort is required. Actually, a deeper knowledge on the topic could offer a different point of view. The aim of the present tutorial is to introduce the experimental design to beginners, by providing the theoretical basic principles as well as a practical guide to use the most common designs, from the experimental plan to the final optimization. Response surface methodology will be discussed, and the main terms related to model computation and statistical evaluations usually performed by software will be explained, in order to give suitable tools to properly use them.

The present paper deals with the multivariate optimization of an extraction-purification strategy for the determination of phytoestrogens (daidzein, genistein, coumestrol, formononetin, and biochanin A) in soy-based meat substitutes by high performance liquid chromatography with tandem mass spectrometry. For a reliable quantitation of these new concerning compounds in such a complex matrix, recovery and matrix effect must be carefully evaluated. Therefore, two sequential experimental designs were used to optimize the sample-pretreatment of soy-based burgers: the chosen technique was the quick, easy, cheap, effective, rugged and safe methodology, which does not require any particular facility or instrumentation. Thanks to the first screening design (Plackett-Burman), the significant factors influencing the studied responses were identified and further investigated through a response surface design (Box-Behnken). The optimal values of the variables (volume of extraction solvent mix/sample mass ratio and two clean-up sorbents) led to quantitative recoveries (97-104%) and low ion suppression (matrix effect 60-93%) for all analytes. This optimized method was characterized by low detection limits (0.2-1.5 ng/g) and excellent intraday precision (RSD 2-4%). It was applied to the determination of the considered compounds in several soy-burgers from the Italian market, detecting low ng/g levels (up to 40 ng/g) of coumestrol, formononetin, and biochanin A, and high concentrations (7.9-78 µg/g) of genistein and daidzein.

The chemical composition of Cannabis sativa L. has been extensively investigated for several years; nevertheless, a detailed lipidome characterization is completely lacking in the literature. To achieve this goal, an extraction and enrichment procedure was developed for the characterization of phospholipids and sulfolipids. Firstly, a study on the solid-liquid extraction was performed, to maximize the recovery of the considered lipids; the best procedure consisted of a simple extraction with a mixture of methanol and chloroform (1:1, v/v). The hemp extracts were analyzed by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry and lipids were tentatively identified by Lipostar. To improve the number of identifications, an enrichment method, based on graphitized carbon black solid phase extraction, was evaluated to fractionate phospholipids and sulfolipids into separate eluates. Recovery and matrix effects of the procedure were determined on a mixture of standard lipids, containing representative compounds for each considered lipid class. The optimized method allowed the tentative identification of 189 lipids, including 51 phospholipids and 80 sulfolipids, in the first and second fractions, respectively. The detection of only 6 sulfolipids in the first fraction and 9 phospholipids in the second fraction proved the efficacy of the fractionation method, which also allowed the number of lipid identifications to be increased compared to the same procedure without enrichment, which scored 100 lipids. Finally, a semi-quantitative analysis permitted the hemp polar lipidome to be characterized. The results of this study allow knowledge of the hemp chemical composition to be improved with a detailed description of its phospho- and sulfolipid profiles. [Figure not available: see fulltext.]

Short peptides, namely di- tri- and tetra peptides, have been proven to play an important diagnostic role in several diseases. Therefore, the development of an analytical approach for their detection and identification is nowadays an important research goal. This paper describes an analytical procedure able to overcome the issues of short peptide isolation, clean-up and identification in plasma samples. Four different protocols were compared and tested to maximize both recovery and total number of identifications of short circulating plasma endogenous peptides. The purified peptides, coming from the four different tested protocols, were separated by zwitterionic hydrophilic liquid chromatography coupled to high-resolution mass spectrometry with the purpose of accomplishing an untargeted investigation based on suspect screening for short peptides in plasma. In particular, the use of Phree(TM) Phospholipid removal cartridge in combination with a purification step by solid phase extraction on a graphitized carbon black sorbent allowed the identification of the largest number of amino acid sequences (91 short peptides). The clean-up procedure allowed to tackle the issue of the low abundance of such peptides and their suppression during mass-spectrometric analysis. The results indicated that sample preparation is therefore fundamental for short peptide analysis in plasma samples.

In this work an optimized GC–MS/MS method, based on a simple and fast derivatization procedure, is proposed for the determination of five phytoestrogens (formononetin, daidzein, coumestrol, genistein and biochanin A) in soy milk. A systematic study of the instrumental analysis conditions was carried out, optimizing different MS acquisition parameters. At the same time, the derivatization procedure was optimized testing three silylating reagents. Among them, N , O -bis-(trimethylsilyl) trifluoroacetamide (BSTFA) was chosen as the most suitable derivatization reagent and an experimental design was performed to find the best conditions for the silylation reaction. The results showed the independence of the derivatization efficiency from the heating treatment applied before analysis, enabling the derivatization directly in the injection port; moreover, the use of a temperature programmed injection enhanced sensitivity and precision of the analysis. The described optimization steps allowed a significant gain in the sensitivity of the overall method, leading to detection limits of 0.1–17.7 μg L − 1 , which are lower or comparable to values reported in the literature for GC–MS analysis of these analytes. High specificity was reached as well, thanks to the careful study of the conditions for tandem MS detection. Precision was good for formononetin, daidzein and coumestrol, with coefficients of variance of 4.7–6.1%; on the contrary, the analysis of genistein and biochanin A was characterized by low precision, probably due to poor stability of the corresponding derivatives. The method was tested on some soy milk samples from the Italian market, proving its suitability for the analysis of phytoestrogens in this complex matrix.

The present work deals with the optimization of a magnetic solid phase extraction of polycyclic aromatic hydrocarbons (PAHs) from sea water, based on molecular imprinted microparticles, for subsequent GC–MS analysis. The extraction efficiency of the magnetic molecular imprinted polymer (MIP) was compared with that of the corresponding non-imprinted polymer; only small differences in the recovery of the extraction procedure were shown, but higher repeatability was observed using MIP. An experimental design (Plackett-Burman) allowed a fast optimization identifying the only significant factor influencing the extraction recovery, namely the ratio between the amount of microparticles and the sample volume. Two optimal ratios, based on the PAHs molecular weight, were selected for 20 mL of sea water samples: 5 mg or 20 mg of microparticles for compounds ranging from 128 to 228 and from 252 to 278 Da, respectively. The recoveries ranges were 76–104% for the low molecular weight PAHs and 95–101% for the high molecular weight PAHs. The analytical method showed satisfactory figures of merit, with detection limits ranging from 30 to 375 ng L−1 and relative standard deviations on replicate extractions from 3.7 to 12.2%. The developed method, which is fast, easy and requires very small volumes of organic solvent, was applied for the determination of PAHs in selected sea water samples.

Microalgae have recently become a popular functional food due to their health benefits. Sulfolipids, a class of substances abundant in this matrix, have been reported to have interesting bioactivities, such as anti-carcinogenic activity. However, despite the potential interest in sulfolipids, a dedicated analytical method for their characterization is currently lacking but would significantly increase the coverage of sulfolipids with respect to the direct lipidomic analysis. To achieve this goal, in this work a procedure, based on graphitized carbon black solid phase extraction, was developed for clean-up and enrichment of sulfolipids (sulfoquinovosyldiacylglycerols and sulfoquinovosylmonoacylglycerols) and it was applied to spirulina (Arthrospira platensis) microalgae. A careful study of the solid phase extraction conditions was performed, first to maximize the recovery of reference standards, then to increase the total number of identified sulfolipids from the spirulina lipid extract. All samples were analysed by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry and lipids were tentatively identified by Lipostar, for a reliable lipid structure assignment. The developed method was compared to the direct lipidomic analysis without enrichment, to establish the enrichment efficiency in terms of number of identifications. From the comparison, the enrichment procedure proved better and allowed the tentative identification of 199 sulfolipids, which is the largest number reported so far for the Arthrospira platensis species. The described method was validated in terms of precision, accuracy, recovery, limit of quantitation and detection for two sulfolipids. Finally, a relative lipid quantitation based on peak area was carried out on the microalgae sample, which indicated nine abundant sulfolipids as representing ca. 60% of sulfolipids in spirulina microalgae.

In this work, an innovative sampling and preconcentration method followed by analysis with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization (LC-ESI-MS/MS) was developed for the determination of different emerging pollutants (five anti-inflammatory drugs and one antibacterial agent) in water matrices. Thin-film microextraction blades, consisting of stainless steel blades with a coating made of divinylbenzene, have been employed. The blades, fixed onto a stainless steel support, were mounted on a laboratory stirrer with adjustable speed, immersed in water samples and eluted with methanol. The analytical procedure was developed, carefully optimizing stirring speed and extraction time. A good reproducibility among the blades was observed; quantitation limits at the ng L-1 level were achieved. Calibration curves were constructed by applying the whole procedure to tap water samples, free from analytes, spiked with standards in the concentration range 0.01-2 μg L-1 ; good linearity was obtained, with R2 between 0.9984 and 0.9991. The optimized method was applied to tap and surface waters; two anti-inflammatory drugs were detected at the ng L-1 level in surface water. In one sample, diclofenac and naproxen were measured at 26 ± 5 and 15 ± 1 ng L-1 , respectively; only diclofenac was quantified in the other sample at 14 ± 3 ng L-1 .

This paper presents the development of an efficient extraction procedure followed by a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of five phytoestrogens (genistein, daidzein, formononetin, biochanin A, and coumestrol) in soy-based meat substitutes. Phytoestrogens are considered endocrine disrupting compounds, and their quantification is important in soy-based products, whose diffusion is increasing nowadays. The HPLC-MS/MS method, with electrospray ionization (ESI) source, was optimized to obtain high specificity and sensitivity, as well as rapidity of the analysis. Three extraction techniques were applied to soy burgers and compared: ultrasound assisted extraction, ultrasound assisted extraction followed by solid phase extraction and the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) methodology. Both ultrasound assisted extraction and QuEChERS proved to be suitable for the determination of phytoestrogens, showing high recoveries, in the range of 86% to 99% and 75% to 105%, respectively. Matrix effect was evaluated, and ion suppression was observed for coumestrol and formononetin, demonstrating the importance of matrix effect assessment when complex samples are analyzed by HPLC-ESI-MS. The complete analytical protocols provided limits of detection and quantitation in soy-burgers at the ng g-1 level for all the considered phytoestrogens. Some soy burger samples were analyzed by both ultrasound assisted extraction and QuEChERS followed by HPLC-MS/MS. High concentration levels of daidzein and genistein (2-59 μg g-1 and 2-72 μg g-1 , respectively) were found; formononetin was in the range of 5 to 26 ng g-1 , while biochanin A and coumestrol were under the limit of quantitation in all samples. The results obtained with the two different sample treatment were in good agreement, proving the precision and accuracy of the described techniques.

Passive sampling and liquid chromatography-tandem mass spectrometry can be profitably employed to detect emerging contaminants in waters at very low concentration levels. In this work, two types of Polar Organic Chemical Integrative Sampler (POCIS) were subjected to calibration at two different temperatures to calculate the sampling rates of eight emerging pollutants (five pharmaceuticals, two perfluorinated compounds and caffeine). Results obtained changing the temperature from + 5 to + 25 °C showed a limited influence on the sampling rate values for all the selected analytes. Preliminary evaluations on storage life-time of POCIS devices were also taken into account. After calibration, samplers were deployed in the inlet and the outlet of two drinking water treatment plants in Northwestern Italy, for two and four weeks; the extracts were then analyzed by means of LC-MS/MS in multiple reaction monitoring mode, which provided high sensitivity and allowed the detection of the selected compounds at the low ng L − 1 level. Three analytes were measured in both treatment plants: the two perfluorinated compounds, in the range 2.93–13.42 ng L − 1 , and caffeine, in the range 0.07–0.93 ng L − 1 .

Recycling and recovering valuable resources from wastewater treatment plants is an important aspect in circular economy. The safe use of sludge and sludge-related products deriving from wastewater treatment strictly depends on their chemical contamination, especially by emerging pollutants. In this work, an analytical method was developed for the determination of a range of selected compounds, included in a recent European watch-list (macrolides, fluoroquinolones, neonicotinoids, carbamates and estrogens), in recovery materials from innovative pilot systems. Both the instrumental analysis by high-performance liquid chromatography-tandem mass spectrometry and the pre-treatment strategy (quick, easy, cheap, effective, rugged and safe technique) were optimized for the purpose. The final method performance were evaluated, revealing determination coefficients (R2) of 0.993–0.9999 for the matrix-matched calibration curves, good accuracy (recovery 68–104% and matrix effect 70–123%), satisfactory precision (relative standard deviation The application of the described method is an essential part of a broader investigation on the suitability and safety of innovative materials coming from waste water treatment plants, in the view of a risk assessment related to their usage.