Assay for urinary estriol during the menstrual cycle based on extraction by a graphitized carbon black cartridge followed by high-performance liquid chromatography

Summary A high-performance liquid chromatography (HPLC) assay for estriol in nonpregnancy urine is described. After Enzymic hydrolysis, the estriol is extracted from urine by the sorbent trap technique utilizing graphitized carbon black (Carbopack B). After some washing steps, estriol is desorbed by a suitable solvent system. After solvent removal, the sample is injected into an HPLC column for estriol quantification. Analytical recovery of estriol was 96.1%. The precision of the method was 2.6 and 4.9% respectively at 145 and 10.6ng/ml of urine. The limit of sensitivity was set at 0.8 ng/ml of urine. The mean contents of estriol in the follicular and luteal phases were respectively 11.3 and 38.8 ng/ml of urine.Dedicated to Prof. Dr. A. Liberti on the occasion of his 70th birthday.     

Fast screening of 88 pharmaceutical drugs and metabolites in whole blood by ultrahigh-performance liquid chromatography–tandem mass spectrometry

Forensic investigations involving acute or lethal intoxication, drug-facilitated sexual assault, driving or workplace impairment frequently require the analysis of fresh or postmortem blood samples to check out a wide variety of pharmaceutical and illicit drugs, even after single-dose consumption. A sensitive and selective ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) screening method was developed for fast screening of 88 psychoactive drugs and metabolites in blood samples, including the ones most frequently involved in acute intoxications and forensic investigations in Italy. The new method allows short sample processing and analysis time (the whole procedure can be accomplished in less than 30 min) together with the simultaneous monitoring of a large number of pharmaceutical substances. These features represent crucial factors in the approach of acute intoxications, when the patient requires urgent and appropriate therapy. Blood sample treatment was limited to protein precipitation. Two UHPLC–MS/MS runs in positive and negative electrospray ionization modes were performed. The data were acquired at unit mass resolution in the selected reaction monitoring mode. According to international guidelines, linearity range, precision, trueness, detection and quantification limits, recovery, selectivity, specificity, carryover, and matrix effect phenomena were determined. Despite the limited sample purification and the inherent decreased chance of eliminating any potential interference, the present multiresidue screening method proved extremely effective and sensitive, allowing the detection of all tested drugs, even those belonging to structurally different classes of substances. Moreover, the developed method is easily susceptible to further expansion to encompass more drugs, either new or those becoming important for criminal investigation. This protocol was also applied to the analysis of authentic blood samples collected from victims of various crimes in routine casework, whose relevance in forensic investigations is presented in five cases.     

A phase I, randomized study of combined IL-2 and therapeutic immunisation with antiretroviral therapy

Background  

Fully functional HIV-1-specific CD8 and CD4 effector T-cell responses are vital to the containment of viral activity and disease progression. These responses are lacking in HIV-1-infected patients with progressive disease. We attempted to augment fully functional HIV-1-specific CD8 and CD4 effector T-cell responses in patients with advanced chronic HIV-1 infection.     

Simultaneous determination of β2‐agonists in human urine by fast‐gas chromatography/mass spectrometry: method validation and clinical application

A fast screening protocol was developed and validated for the simultaneous determination of 15 β₂‐agonists in human urine (bambuterol, cimbuterol, clenbuterol, fenoterol, formoterol, isoproterenol, mapenterol, metaproterenol, procaterol, ractopamine, ritodrine, salbutamol, salmeterol, terbutaline, tulobuterol). The overall sample processing includes deconjugation with enzyme hydrolysis, liquid–liquid extraction, followed by derivatization of the extract and detection of β₂‐agonists trimethylsilyl‐derivatives by fast‐gas chromatography/electron impact–mass spectrometry (fast‐GC/EI‐MS). Sample extraction and derivatization were optimized with the purpose of improving recoveries and reaction yields for a variety of analytes with different structures simultaneously, while keeping the procedure simple and reliable. Validation parameters were determined for each analyte under investigation, including selectivity, linearity, intra‐ and inter‐assay precision, extraction recoveries and signal to noise ratio (S/N) at the lowest calibration level. Fast‐GC/MS sequences, based on the use of short columns, high carrier‐gas velocity and fast temperature ramping, allow considerable reduction of the analysis time (7 min), while maintaining adequate chromatographic resolution. The overall GC cycle time was less than 9 min, allowing a processing rate of 6 samples/h. High MS‐sampling rate, using a benchtop quadrupole mass analyzer, resulted in accurate peak shape definition under both scan and selected ion monitoring modes, and high sensitivity in the latter mode. The method was successfully tested on real samples arising from clinical treatments. Copyright © 2009 John Wiley & Sons, Ltd.     

A rapid method based on hot water extraction and liquid chromatography-tandem mass spectrometry for analyzing tetracycline antibiotic residues in cheese

A rapid, specific, and sensitive procedure for determining residues of 4 widely used tetracycline antibiotics and 3 of their 4-epimers in cheese is presented. The method is based on the matrix solid-phase dispersion (MSPD) technique followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). After dispersing samples of mozzarella, asiago, parmigiano, gruyere, emmenthal, and camembert on sand, target compounds were eluted from the MSPD column by passing through it 6 mL water heated at 70 degrees C. After acidification and filtration, 200 microL of the aqueous extract was directly injected into the LC column. For analyte identification and quantification, MS data acquisition was performed in the multireaction monitoring mode, selecting 2 precursor ion-to-product ion transitions for each target compound. Hot water appeared to be an efficient extractant, because absolute recoveries were no lower than 78%. Using demeclocycline as a surrogate analyte, recoveries of analyte added to the 6 types of cheeses at the 30 ng/g level were 96-117%, with relative standard deviation (RSD) not higher than 9%. Statistical analysis of the mean recovery data showed that the extraction efficiency was not dependent on the type of cheese analyzed. This result indicates that this method could be applied to other cheese types not considered here. At the lowest concentration considered, i.e., 10 ng/g, the accuracy of the method ranged between 90 and 107%, with RSDs not larger than 12%. Based on a signal-to-noise ratio of 10, limits of quantitation were estimated to be 1-2 ng/g.     

Simultaneous analysis of several synthetic cannabinoids,THC, CBD and CBN,in hair by ultra‐high performance liquid chromatography tandem mass spectrometry. Method validation and application to real samples

A simple procedure for the quantitative detection of JWH‐018, JWH‐073, JWH 200, JWH‐250, HU‐210, Δ⁹‐tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in hair has been developed and fully validated. After digestion with NaOH and liquid–liquid extraction, the separation was performed with an ultra‐high performance liquid chromatography system coupled to a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. The absence of matrix interferents, together with excellent repeatability of both retention times and relative abundances of diagnostic transitions, allowed the correct identification of all analytes tested. The method was linear in two different intervals at low and high concentration, with correlation coefficient values between 0.9933 and 0.9991. Quantitation limits ranged from 0.07 pg/mg for JWH‐200 up to 18 pg/mg for CBD The present method for the determination of several cannabinoids in hair proved to be simple, fast, specific and sensitive. The method was successfully applied to the analysis of 179 real samples collected from proven consumers of Cannabis, among which 14 were found positive to at least one synthetic cannabinoid. Copyright © 2012 John Wiley & Sons, Ltd.