Eugenia Gallardo

Strategies to improve the solubility and stability of stilbene antioxidants: A comparative study between cyclodextrins and bile acids

Aiming at the development of an active food packaging, the goal of this study was to increase stilbenes (resveratrol (RV), pterostilbene (PT) and pinosylvin (PS)) aqueous solubility and stability using hydropropyl-cyclodextrins (HP-CDs) and bile salts. To evaluate stilbene concentration, an HPLC-DAD method was validated. Stilbene solubility was improved by the formation of inclusion complexes and micellar systems with higher solubility values obtained for the inclusion complexes with cyclodextrins. Inclusion complexes revealed a 1:1 stoichiometry for RV and PT and a 1:2 for PS. Solid state characterisation was carried out using X-ray diffraction, Fourier transform infrared spectroscopy and differential scanning calorimetry. (1)H NMR studies were also performed to characterise the prepared complexes. Photostability studies revealed that CDs were able to increase stilbene photostability at 4 °C. This work showed that stable stilbene solutions can be achieved using hydroxypropyl-CDs, contributing for their incorporation in several materials for the food and pharmaceutical industries.

Hair: a complementary source of bioanalytical information in forensic toxicology

Hair has been used for years in the assessment and documentation of human exposure to drugs, as it presents characteristics that make it extremely valuable for this purpose, namely the fact that sample collection is performed in a noninvasive manner, under close supervision, the possibility of collecting a specimen reflecting a similar timeline in the case of claims or suspicion of a leak in the chain of custody, and the increased window of detection for the drugs. For these reasons, testing for drugs in hair provides unique and useful information in several fields of toxicology, from which the most prominent is the possibility of studying individual drug use histories by means of segmental analysis. This paper will review the unique role of hair as a complementary sample in documenting human exposure to drugs in the fields of clinical and forensic toxicology and workplace drug testing.

Combinatorial delivery of Crizotinib–Palbociclib–Sildenafil using TPGS-PLA micelles for improved cancer treatment

The co-delivery of multiple chemotherapeutics by micellar delivery systems is a valuable approach to improve cancer treatment since various disease hallmarks can be targeted simultaneously. However, the delivery of multiple drugs requires a nanocarrier structure that can encapsulate various bioactive molecules. In this study, we evaluate the simultaneous encapsulation of a novel triple drug combination in D-α-tocopheryl polyethylene glycol 1000 succinate-poly(lactic acid) (TPGS-PLA) amphiphilic micelles for cancer therapy. The drug mixture involves two anti-tumoral drugs, Crizotinib and Palbociclib combined with Sildenafil, a compound that is capable of increasing drug accumulation in the intracellular compartment. Such combination aims to achieve an enhanced cytotoxic effect in cancer cells. Our results demonstrated that TPGS-PLA copolymers self-assembled into stable nanosized micelles (158.3nm) capable of co-encapsulating the three drugs with high loading efficiency. Triple drug loaded TPGS-PLA micelles were internalized in A549 non-small lung cancer cells and exhibited an improved cytotoxic effect in comparison with single (Crizotinib) or dual (Crizotinib-Palbociclib) drug loaded micelles, indicating the therapeutic potential of the triple co-delivery strategy. These findings demonstrate that TPGS-PLA micelles are suitable carriers for multiple drug delivery and also that this particular drug combination may have potential to improve cancer treatment.

Bioanalytical procedures and recent developments in the determination of opiates/opioids in human biological samples

The use and abuse of illegal drugs affects all modern societies, and therefore the assessment of drug exposure is an important task that needs to be accomplished. For this reason, the reliable determination of these drugs and their metabolites in biological specimens is an issue of utmost relevance for both clinical and forensic toxicology laboratories in their fields of expertise, including in utero drug exposure, driving under the influence of drugs and drug use in workplace scenarios. Most of the confirmatory analyses for abused drugs in biological samples are performed by gas chromatographic–mass spectrometric methods, but use of the more recent and sensitive liquid chromatography–(tandem) mass spectrometry technology is increasing dramatically. This article reviews recently published articles that describe procedures for the detection of opiates in the most commonly used human biological matrices, blood and urine, and also in unconventional ones, e.g. oral fluid, hair, and meconium. Special attention will be paid to sample preparation and chromatographic analysis.

Current technologies and considerations for drug bioanalysis in oral fluid.

Drug oral fluid analysis was first used almost 30 years ago for the purpose of therapeutic drug monitoring. Since then, oral fluid bioanalysis has become more popular, mainly in the fields of pharmacokinetics, workplace drug testing, criminal justice, driving under the influence testing and therapeutic drug monitoring. In fact, oral fluid can provide a readily available and noninvasive medium, without any privacy loss by the examinee, which occurs, for instance, during the collection of urine samples. It is believed that drug concentrations in oral fluid may parallel those measured in blood. This feature makes oral fluid an alternative analytical specimen to blood, which assumes particular importance in roadside testing, the most published application of this sample. Great improvements in the development of accurate and reliable methods for sample collection, in situ detection devices (on-site drug detection kits), and highly sensitive and specific analytical methods for oral fluid testing of drugs have been observed in the last few years. However, without mass spectrometry-based analytical methods, such as liquid chromatography coupled to mass spectrometry (LC-MS) or tandem mass spectrometry (LC-MS/MS), the desired sensitivity would not be met, due to the low amounts of sample usually available for analysis. This review will discuss a series of published papers on the applicability of oral fluid in the field of analytical, clinical and forensic toxicology, with a special focus on its advantages and drawbacks over the normally used biological specimens and the main technological advances over the last decade, which have made oral fluid analysis of drugs possible.

Development and validation of an analytical method for the determination of trans- and cis-resveratrol in wine: analysis of its contents in 186 Portuguese red wines.

A simple procedure based on solid-phase extraction and high performance liquid chromatography coupled to diode array detector has been developed and validated for the qualitative and quantitative analysis of cis- and trans-resveratrol in wines. The method was linear from 0.025 (lower limit of quantitation, LLOQ) to 15 μg/mL for trans-resveratrol and from 0.023 (LLOQ) to 0.92 μg/mL for cis-resveratrol, with correlation coefficients higher than 0.99 for both isomers. Intra- and interday precision and accuracy were in conformity with the criteria normally accepted in method validation, that is, CVs inferior to 15% and mean relative errors within a ±14% interval. The extraction presented mean efficiencies close to 100% for both analytes. The validated methodology was applied to 186 Portuguese red wines from different regions, grape varieties and vintage. The results obtained showed that the content of trans-resveratrol in red wines ranged from 0.05 to 10.9 μg/mL, while the concentrations of cis-resveratrol ranged from 0.04 to 8.71 μg/mL.

Determination of piperazine-type stimulants in human urine by means of microextraction in packed sorbent and high performance liquid chromatography-diode array detection

A method using microextraction by packed sorbent (MEPS) and high performance liquid chromatography-diode array detection (HPLC-DAD) is described for the determination of piperazine-type stimulants in human urine. The studied compounds were 1-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl) piperazine (TFMPP), 1-(3-chlorophenyl) piperazine (mCPP) and 1-(4-methoxyphenyl) piperazine (MeOPP); 1-(2-chlorophenyl)-piperazine (oCPP) was used as internal standard (IS). The factors which might influence the extraction were screened previously using the fractional factorial design approach, and none of them influenced significantly the process. The procedure was linear for concentrations ranging from 0.1 (lower limit of quantitation--LLOQ) to 5 μg/mL, with determination coefficients (R(2)) higher than 0.99 for all analytes in all runs. The limits of detection were 0.1 μg/mL for BZP and TFMPP, while for MeOPP and mCPP 0.05 μg/mL was obtained. Intra- and interday precision ranged from 1 to 14%, and accuracy was within a ± 15% interval for all analytes, fulfilling the criteria normally accepted in bioanalytical method validation. Under the optimized conditions, extraction efficiency was higher than 80% for all analytes, except BZP (50%). MEPS showed to be a rapid (<2 min) and simple procedure for the determination of piperazine-type stimulants in human urine, allowing reducing the handling time and costs usually associated to this type of analysis. Furthermore, the fact that only 0.1 mL of sample is required make this method a valuable and powerful tool for drug monitoring in human urine in situations where those compounds are involved, for instance in forensic scenarios.

Rapid determination of piperazine-type stimulants in human urine by microextraction in packed sorbent after method optimization using a multivariate approach

This paper describes the analysis of piperazine-type stimulants [1-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)piperazine (TFMPP), 1-(3-chlorophenyl)piperazine (mCPP) and 1-(4-methoxyphenyl)piperazine (MeOPP)] in low volume urine samples (0.1 mL) by microextraction in packed sorbent and liquid chromatography-diode array detection. Analyte extraction has been comprehensively optimized, and the influencing factors were screened by means of the fractional factorial design approach. Several parameters susceptible of influencing the process were studied, and these included extraction sorbent type (C(8) and C(18)), sample dilution (1:2 and 1:4), number of aspirations through the device (2 and 8) and the amount of methanol on both the washing (0 and 10%) and eluting solvents (10 and 100%). The method was linear from 0.5 (lower limit of quantitation) to 5 μgmL(-1), with determination coefficients higher than 0.99 for all compounds. Intra- and interday precision ranged from 1 to 9%, trueness was within a ± 11% interval for all analytes, and analyte recoveries were of about 70% for mCPP and TFMPP, and of about 10% for MeOPP and BZP. The method has shown to be selective, as no interferences from endogenous substances were detected by analysis of blank samples, and the analytes were stable in the samples for short periods at room temperature, after three freeze/thaw cycles and in processed samples. Due to its simplicity and speed, this method can be successfully applied in the screening and quantitation of these compounds in urine samples, and is suitable for application in forensic toxicology routine analysis.

LC-MS: a powerful tool in workplace drug testing

Workplace drug testing is a well-established application of forensic toxicology and it aims to reduce workplace accidents caused by affected workers. Several classes of abused substances may be involved, such as alcohol, amphetamines, cannabis, cocaine, opiates and also prescription drugs, such as benzodiazepines. The use of alternative biological specimens such as hair, oral fluid or sweat in workplace drug testing presents several advantages over urinalysis-mainly the fact that sample collection can be performed easily without infringing on the examinees privacy, so the subject is more likely to perform the test. However, drugs are usually present in these alternative specimens at low concentrations and the amount of sample available for analysis is small. The use of highly sensitive techniques is therefore necessary. In fact, the successful interface of liquid chromatography with mass spectrometry (LC-MS) has brought a new light into bioanalytical and forensic sciences as it allows the detection of drugs and metabolites at concentrations that are difficult to analyse using the more commonly adopted GC-MS based techniques. This paper will discuss the importance of LC-MS in supporting workplace drug-testing programmes. The combination of LC-MS with innovative instrumentation such as triple quadrupoles, ion traps and time-of-flight mass spectrometers will also be focused.

Analytical approach to determine biogenic amines in urine using microextraction in packed syringe and liquid chromatography coupled to electrochemical detection.

The goal of this work was to develop and validate an analytical method for the detection and quantification of the biogenic amines serotonin (5-HT), dopamine (DA) and norepinephrine (NE), using microextraction in packed syringe (MEPS) and liquid chromatography coupled to electrochemical detection (HPLC-ED) in urine. The method was validated according to internationally accepted guidelines from the Food and Drug Administration. Linearity was established between 50 and 1000 ng/mL for 5-HT and between 5 and 1000 ng/mL for DA and NE, with determination coefficients (R(2)) >0.99 for all compounds. The limits of quantification and detection were respectively 50 and 20 ng/mL for 5-HT, and 5 and 2 ng/mL for DA and NE. Within- and between-run precision ranged from 0.84 to 9.41%, while accuracy ranged from 0.79 to 12.76% for all compounds. The intermediate precision and accuracy were 1.50-8.36 and 0.54-13.51%, respectively. The method was found suitable for clinical routine analysis of the studied compounds, using a sample volume of 0.5 mL. This is the first study employing a commercially available MEPS column for the simultaneous detection and quantification of 5-HT, DA and NE in urine by coulometric detection.